( F ) unwinding assays upon Ago3 - piRISC - dependent target cleavage as in ( D ) . Recombinant DDX43 was added incubated in reaction mixture upon cleavage for 0 min ( i . e . , no cleavage ) , 15 min , 30 min , 60 min , materials were separated using a magnet to supernatant ( Sup . ) bead ( Beads ) fractions . RNAs by Ago3 are shown as " " . DDX43 fails to unwind RNAs from Siwi - piRISC . − DDX43 means no DDX43 added in reaction mixture .

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(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.


( C ) Immunoprecipitation of Ago3 with Flag - tagged DDX43 WT its D399A E400Q mutants . Ago3 DDX43 were detected by western blotting using anti - Ago3 anti - Flag - antibodies , respectively . Flag - EGFP was used as a negative control . E400Q mutation in DDX43 impairs interaction between Ago3 DDX43 . IgG h . c . : IgG heavy chain .

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(C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain.


( F ) unwinding assays upon Ago3 - piRISC - dependent target cleavage . Unwinding activities of DDX43 WT its D399A , E400Q , ∆ KH mutants are shown . RNAs by Ago3 are shown as " " .

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(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA".


( A ) Masson ' s trichrome representative of pulmonary fibrosis .

(A) Masson's trichrome representative images of pulmonary fibrosis and quantification.


( B ) Relative transcript expression of Fibronectin , ( C ) Collagen I ( D ) Collagen III to GAPDH .

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(B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH.


( E ) Masson ' s trichrome representative of vascular wall thickening .

(E) Masson's trichrome representative images of vascular wall thickening and quantification.


( F ) Masson ' s trichrome representative correlation between lung fibrosis vascular wall thickness in PF PF - PH . Out of 14 PF PF - PH , 3 of them had no non - fibrotic therefore were excluded for comparison between non - fibrotic fibrotic areas ( n = 11 / group ; Ashcroft score non - fibrosis : 0 - 2 ; mild - fibrosis : 3 - 5 ; severe - fibrosis > 5 ) ( * compared to non - fibrotic areas of same group of ) .

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(F) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients).


( G ) Masson ' s trichrome representative of vascular wall thickening in non - fibrotic ( Ashcroft score 0 - 2 ) fibrotic ( Ashcroft score > 2 ) in PF PF - PH . number of samples per group for each experiment are written within each bar graph .

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(G) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph.


( A ) Representative of Ki67 - positive pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC , aSMA in ) ( * vs PF , * p < 0 . 05 , * * * p < 0 . 001 ) .

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(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001).


Relative expression of Slug mRNA ( B ) normalized to GAPDH in PF PF - PH .

Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Slug ( C ) normalized to GAPDH in PF PF - PH .

Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients.


( D ) Representative of Slug ( red ) expression in bronchial epithelial ( EpC ) , fibroblasts ( Fib ) ( vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial ( EC , von Willebrand Factor in green ) pulmonary vascular smooth muscle ( SMC , aSMA in ) ; .

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(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.


( E ) Relative CD68 mRNA expression normalized to

(E) Relative CD68 mRNA expression normalized to GAPDH


( F ) Representative of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic fibrotic areas of lung in PF PF - PH . number of samples per group for each experiment are included within each bar graph .

(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.


( A ) of 20 genes up - regulated in GSE24988 microarray known to be Slug transcriptionnal target .

(A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target.


( C ) of seven transcriptional targets of Slug known to promote proliferation to be extracellular .

(C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular.


Relative expression of Prolactin - induced ( PIP ) mRNA ( D ) normalized to GAPDH in PF PF - PH .

Relative expression of Prolactin-induced protein (PIP) mRNA (D) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Prolactin - induced ( PIP ) expression normalized to GAPDH in PF PF - PH .

Relative expression of Prolactin-induced protein (PIP) expression normalized to GAPDH in PF and PF-PH patients.


( F ) Representative of PIP immunohistochemistry showing its extracellular localization .

(F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification.


Proliferation on healthy fibroblast ( G ) Pulmonary arterial EC ( H ) on healthy pulmonary arterial SMC ( I in presence or absence of PIP . number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


Proliferation on healthy pulmonary arterial SMC J ) in presence or absence of PIP . number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy pulmonary arterial SMC J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


( A ) Masson ' s trichrome representative of lung fibrosis two after PBS or bleomycin instillation .

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(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.


of ( B ) fibronectin , ( C ) Collagen I ( D ) Collagen III mRNA .

Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA.


( F ) Right ventricular systolic pressure ( RVSP ) ( G ) Right ventricular hypertrophy index following experimental protocol .

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(F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol.


( H ) Masson ' s trichrome representative of Ashcroft score in experimental model .

(H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model.


Relative expression of fibronectin ( I ) , collagen I ( J ) collagen III ( K ) mRNA normalized to GAPDH .

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Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH.


( L ) Masson ' s trichrome representative of vascular remodeling .

(L) Masson's trichrome representative images and quantification of vascular remodeling.


( M ) Masson ' s trichrome representative of vascular remodeling between fibrotic non - fibrotic areas in experimental model . number of samples per group for each experiment are included within each bar graph .

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(M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph.


( A ) Representative of Ki67 - positive ( red ) pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC ) in experimental model .

(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model.


Relative expression of Slug mRNA ( B ) normalized to GAPDH .

Relative expression of Slug mRNA (B) normalized to GAPDH.


Relative expression of Slug ( C ) normalized to GAPDH .

Relative expression of Slug protein (C) normalized to GAPDH.


( D ) Representative of Slug ( red ) expression in bronchial epithelial ( EpC ) , fibroblasts ( Fib , vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial ( EC , vWF ) smooth muscle ( SMC , aSMA in ) ; . ( * vs Bleo , p < 0 . 05 )

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(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)


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(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats


( F ) Representative of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic fibrotic areas of lung in in Bleo Bleo - MCT rats . number of samples per group for each experiment are included within each bar graph .

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(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of PIP mRNA ( A ) normalized to GAPDH .

Relative expression of PIP mRNA (A) normalized to GAPDH.


Relative expression of PIP ( B ) normalized to GAPDH .

Relative expression of PIP protein (B) normalized to GAPDH.


( C ) Representative PIP . number of samples per group for each experiment are included within each bar graph .

(C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of Slug ( B ) normalized to GAPDH .

Relative expression of Slug protein (B) normalized to GAPDH.


Relative expression of Slug mRNA ( C ) normalized to GAPDH .

Relative expression of Slug mRNA (C) normalized to GAPDH.


( D ) right ventricular systolic pressure ( RVSP ) ( E ) right ventricular hypertrophy index in PF - PH rats treated with Si - Slug compared to Si - Scrm .

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(D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm.


Relative expression of fibronectin ( F ) , Collagen I ( G ) Collagen III ( H ) normalized to GAPDH .

Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH.


( I ) Masson ' s trichrome representative of Ashcroft score . ( J ) Masson ' s trichrome representative of vascular wall thickness .

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(I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness.


( K ) Masson ' s trichrome representative of vascular remodeling between non - fibrotic fibrotic areas of lung .

(K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung.


( L ) Representative of Ki67 - positive ( red ) pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC , aSMA in ) . number of samples per group for each experiment are included within each bar graph .

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(L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.


( A ) Representative of Slug - positive macrophages ( slug , red ; CD68 green ) .

(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green).


( B ) Relative expression of CD68 mRNA normalized to GAPDH .

(B) Relative expression of CD68 mRNA normalized to GAPDH.


( C ) Representative of Slug - positive macrophages between non - fibrotic fibrotic areas of lung .

(C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung.


Relative expression of PIP mRNA ( D ) normalized to GAPDH .

Relative expression of PIP mRNA (D) normalized to GAPDH.


Relative expression of PIP ( E ) normalized to GAPDH .

Relative expression of PIP protein (E) normalized to GAPDH.


( F ) Representative of PIP immunohistochemistry .

(F) Representative images and quantification of PIP immunohistochemistry.


E ) H2AQ105me H3K56ac in deletions of genes important for H3K56ac

E) H2AQ105me and H3K56ac levels in deletions of genes important for H3K56ac deposition


G - J ) ChIP - qPCR for indicated antibodies after treatment with 5nM rapamycin for indicated time points . Samples were either normalized to input or to histone . n = 3 , biological replicates , error bars are standard error of mean . * , p < 0 . 05 , relative to t = 0 , on unpaired , 2 - t -

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G-J) ChIP-qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points. Samples were either normalized to input or to the core histone. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, relative to t=0, based on unpaired, 2-tailed t-test


B ) Volcano plot of quantitative SILAC experiments . Left upper quadrant indicates significantly , 2 - fold enriched bound specifically to H2AQ105me . Members of FACT ( SPT16 / POB3 ) are highlighted in red .

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B) Volcano plot of quantitative SILAC experiments. Left upper quadrant indicates significantly, 2-fold enriched proteins bound specifically to H2AQ105me. Members of the FACT complex (SPT16/POB3) are highlighted in red.


C ) Peptide pulldown using recombinantly purified Nhp2 . Eluted peptides were slot blotted detected using streptavidine - HRP .

C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were slot blotted and detected using streptavidine-HRP.


E ) ChIP - seq profile of Nhp2 ( orange ) its respective input ( black ) across entire chromosome XII of yeast . H2A - normalised H2AQ105me ( grey ) was directly taken as comparison . Magnification shows two annotated rDNA loci in yeast genome indicated by black bars below tracks . Scale bar is for magnification .

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E) ChIP-seq profile of Nhp2 (orange) and its respective input (black) across the entire chromosome XII of yeast. H2A-normalised H2AQ105me (grey) was directly taken as comparison. Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks. Scale bar is for the magnification.


D ) Proteomic of Nhp2 interactors . Highlighted in red are known members of H / ACA pseudouridylation . red dashed indicates significance

D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoff


G ) Peptide pulldowns from human ( HEK293T ) to for conservation of reported interactions between Nhp2 members of SSU processome on H2AQ105me . Dyskering ( Cbf5 homolog ) was probed to for presence of members of H / ACA . Pulldown efficiency was analyzed by western blotting using indicated antibodies .

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G) Peptide pulldowns from human cells (HEK293T) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me. Dyskering (Cbf5 homolog) was probed to test for the presence of members of the H/ACA complex. Pulldown efficiency was analyzed by western blotting using the indicated antibodies.


I ) of Northern Blot results . Normalised to GAPDH , n = 3 , error bars are standard error of mean . Asterix indicates p - value ( * , p < 0 . 05 , on unpaired , 2 - t - ) .

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I) Quantification of Northern Blot results. Normalised to GAPDH, n=3, error bars are standard error of the mean. Asterix indicates p-value (*, p<0.05, based on unpaired, 2-tailed t-test).


E . Normalized ThT kinetics of 10μM Biot - Aβ1 - 42 with timepoints of samples that incubated with Aβ1 - 42 membranes .

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E. Normalized ThT kinetics of 10μM Biot-Aβ1-42 with timepoints of samples that incubated with Aβ1-42 membranes.


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I. ThT kinetics of Biot-Aβ1-42 seeding. 10μM of Biot-Aβ1-42 incubated with 0.5 or 1 μM of Biot-Aβ1-42 seeds.


D . Binding of Biot - Aβ1 - 42 to homologue peptides derived from ~ 520 randomly selected .

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D. Binding of Biot-Aβ1-42 to homologue peptides derived from ~520 randomly selected proteins.


E . Summary of Biot - Aβ1 - 42 binding throughout 8 membranes , color indicates mean between membranes of outline standard deviation .

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E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.


A - C . ThT kinetics of 10μM rAβ1 - 42 alone or in presence ( 1 : 1 ) of 3 homologue peptides ( full screen on Appendix Figure S3 , S4 , n = 2 independent experiments with 4 repeats ) . Reused in Appendix figure

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A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3


I . Representative AFM height of Aβ fibrils alone or in a 1 : 1 mixture with P3 , P5 , P8 P12 peptides are show in top row . boxes indicate magnified regions shown in second row . Arrows indicate locations of representative fibrils shown in magnified detail , each shown as a 200 nm digitally straightened segment a 100 nm segment of corresponding 3D surface envelope model that was calculated from image data . scale bar for each row are shown to left , with both 3D model straightened image data representing 10 nm . colour scale of 3D models from blue to yellow indicate ( from low to high ) between fibril surface fibril centre axis to demonstrate their twist patterns . average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single , not fragmented , amyloid fibrils are shown in bottom row .

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I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.


A . Overrepresentation of Aβ APRs in amyloid plaques of AD brains ( statistics : hypergeometric with Bonferroni correction ) * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . original data was taken from Xiong et al included three biological replicates .

A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.


G . Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains . ( statistics : hypergeometric with Bonferroni correction ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 original data was taken from Xiong et al included three biological replicates .

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G. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.


A . Overrepresentation of Aβ APRs in amyloid plaques of APP / PS1 mouse brains ( mean ± SD ) from two biological replicates . ( statistics : hypergeometric with Bonferroni correction )

A. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)


D . No overrepresentation of Aβ APRs was observed in from Glial inclusions ( α - synuclein ) . ( statistics : hypergeometric with Bonferroni correction )

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D. No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions (α-synuclein aggregates). (statistics: hypergeometric test with Bonferroni correction)


D . Representative of 3 that can induce aggregation of Aβ1 - 42 in biosensor . Increase aggregation is observed in expressing construct but not GFP alone ( left panels ) . Scale bar : 100μm . FRAP of resulting shows no recovery ( in detail at Appendix Figure S11 ) . Scale bar : 2 . 5μm Removal of homologue regions resulting in reduced aggregation ( right panels ) . ( n = 3 independent experiments ) .

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D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).


E . of number of spots per in expressing / not expressing construct ( identified by GFP , transfection reporter ) ( n = 3 independent experiments , statistics : ordinary one - way Anova with Dunnett T3 multiple comparison correction , unpaired t - for transfected / non - transfected ) . Bar plot : mean with 95 % CI . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 .

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E. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001


F - G . of Spots per for construct6 construct6 control ( 6Ctrl , removal of homologue region ) ( F ) construct10 construct10 control ( 10ctrl , removal of homologue region ) ( G ) . ( n = 4 independent experiments , statistics : ordinary one - way Anova ) . Bar plot : mean with 95 % CI . * * * * P ≤ 0 .

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F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001


H - I ) Confocal of colocalization of of interest with Aβ . ( Contrast of were enhanced to 0 . 1 % saturated pixels using Fiji ) . Scale bars :

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H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm


( A ) immunoprecipitation ( ChIP ) of receptor binding to ER8 element ( region 1 ) of nrf2 promoter . Immunoprecipitation with nonspecific IgG PCR amplification of an adjacent region ( 2 ) served as controls . ChIP assays were also carried out to analyse binding to ER8s in il10 , ddit3 tyrobp genes . RAR , retinoic acid receptor ; VDR , vitamin D receptor .

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

(A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor.


( B ) Reverse transcription - PCR of 1 , 25 ‐ dihydroxyvitamin D3 ( 1 , 25D3 ) ‐ or retinoic acid ( RA ) ‐ regulated expression of ddit3 , il10 , tyrobp in U937 , Calu ‐ 3 MCF ‐ 7 .

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

(B) Reverse transcription-PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.


( A ) Electrophoretic mobility shift ( EMSA ) of binding of vitamin D receptors ( VDRs ) / retinoid X receptors ( RXRs ) to thep19ink4dER8 , with binding to mouseosteopontin ( mop ) DR3vitamin D responsive element as a control , along with a comparison of binding to ER6 ER8 motifs ( right ‐ panel ) .

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

(A) Electrophoretic mobility shift assay (EMSA) of binding of vitamin D receptors (VDRs)/retinoid X receptors (RXRs) to thep19ink4dER8, with binding to the mouseosteopontin (mop) DR3vitamin D responsive element as a control, along with a comparison of binding to ER6 and ER8 motifs (right‐hand panel).


( B ) EMSA of retinoic acid receptor ( RAR ) / RXR binding to p19ink4d ER8 , rarβ DR5 retinoic acid response element , as a control .

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

(B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control.


( C ) immunoprecipitation ( ChIP ) of binding of VDRs RARs to p19ink4d ER8 ( region 1 ) polymerase II ( POLII ) binding to transcription start site .

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(C) Chromatin immunoprecipitation (ChIP) analysis of binding of VDRs and RARs to the p19ink4d ER8 (region 1) and RNA polymerase II (POLII) binding to the transcription start site.


( D ) ReChIP confirms presence of VDR / RXRs RAR / RXRs but not of VDR / RARs on p19ink4d ER8 .

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(D) ReChIP analysis confirms the presence of VDR/RXRs and RAR/RXRs but not of VDR/RARs on the p19ink4d ER8.


( E ) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter . 3 ( 1 , 25D3 ) ‐ ( F ) retinoic acid ( RA ) ‐ regulated ( G ) luciferase expression is dependent on ER8 element in p19ink4d promoter . Luciferase expression driven by a thymidine kinase ( tk ) control promoter is shown .

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

(E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.


3 retinoic acid induce p19ink4d expression . Expression of p19ink4d messenger by reverse transcription - PCR ( A ) or by western blotting ( B ) was carried out on extracts of treated with 3 ( 1 , 25D3 ) or retinoic acid ( RA ) , as indicated .

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

3 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription-PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 3 (1,25D3) or retinoic acid (RA), as indicated.


( C ) Treatment with 1 , 25D3 or RA transiently enhances co ‐ immunoprecipitation ( IP ) of p19INK4D with cyclin ‐ dependent kinase CDK4 in U937 . Similar results were obtained in SCC25 ( not shown ) . A control western blot ( W ) of CDK4 in ligand ‐ treated is shown below .

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

(C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase CDK4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below.


( D ) Treatment with 1 , 25D3 enhances co ‐ immunoprecipitation of p27KIP1 with CDK4 in U937 . Extracts of U937 were probed for p27KIP1 CDK4 , immunoprecipitated with an anti ‐ CDK antibody probed for p27KIP1 co ‐ immunoprecipitation .

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

(D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.


( A ) Short interfering RNAs ( siRNA ) knockdown of p19ink4d messenger in SCC25 . SiRNAs were directed against p19ink4d , scrambled p19ink4d ( Scr . ) or cyclophilin ( cyc . ) controls .

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(A) Short interfering RNAs (siRNA) knockdown of p19ink4d messenger RNA and protein in SCC25 cells. SiRNAs were directed against p19ink4d, scrambled p19ink4d (Scr.) or cyclophilin (cyc.) controls.


( B ) Combined knockdown of p27KIP1 p19INK4D in SCC25 analysed by western blotting for p27KIP1 ( top ) p19INK4D ( bottom ) .

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(B) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 (top) and p19INK4D (bottom).


( C , D ) ‐ activated sorting of ‐ cycle distribution of control SCC25 , or transfected with scrambled ( Scr ) , p19ink4d ‐ or p27kip1 ‐ directed siRNAs , individually or together ( Dbl ) . ( C ) were treated with vehicle or 1 , 25 ‐ dihydroxyvitamin D3 ( 1 , 25D3 ) , as indicated . ( D ) were treated with DMSO vehicle , retinoic acid ( RA ) or 1 , 25D3 , as indicated .

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

(C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated.


( E ) Loss of p19 enhances lysosomal β ‐ galactosidase activity in SCC25 . ( Left ) Histogram of numbers of for β ‐ galactosidase activity in absence ( − ) or presence of 1 , 25D3 in control ( − ) or in transfected with scrambled ( Scr ) or p19 siRNAs . ( Right ) Bright field of untransfected ( − / − ) or p19INK4D ‐ depleted treated with 1 , 25D3 ( p19 / D3 ) .

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

(E) Loss of p19 enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19 siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3).


( F ) Loss of p19INK4D induces lysosomal fusion . of lysosomal fusion ( autophagosomes ) in SCC25 treated as in ( E ) , stained for incorporation of lysotracker red . Nuclei were counterstained with Hoechst ( blue ) .

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(F) Loss of p19INK4D induces lysosomal fusion. Analysis of lysosomal fusion (autophagosomes) in SCC25 cells treated as in (E), stained for incorporation of lysotracker red. Nuclei were counterstained with Hoechst (blue).


A . ROCK2 immunohistochemistry stained sections of normal stages I , II III cases of human pancreas adenocarcinoma . Scale bar = 50 µm ( left ) . Histoscores of ROCK2 ( right ) in normal pancreas ( n = 5 ) , pancreas adenocarcinoma stage I ( n = 22 ) , stage II ( n = 46 ) stage III / IV ( n = 4 ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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A. ROCK2 immunohistochemistry stained sections of normal and stages I, II and III cases of human pancreas adenocarcinoma. Scale bar = 50 µm (left). Histoscores of ROCK2 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma stage I (n=22), stage II (n=46) and stage III/IV (n=4). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


B . Overall survival of 107 without alterations versus 37 with ROCK1 / or ROCK2 gene amplification , significantly increased mRNA or truncation mutation from TCGA research network . Survival p value determined by Log - Rank .

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B. Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and/or ROCK2 gene amplification, significantly increased mRNA or truncation mutation from TCGA research network. Survival p value determined by Log-Rank test.


C , D . Log2 median - centered ROCK1 or ROCK2 expression in normal ( n = 39 ) vs PDAC ( n = 39 ) , normal ( n = 5 ) vs pancreatic adenocarcinoma ( PAC ) ( n = 12 ) , or normal ( n = 6 ) vs pancreatic carcinoma ( PC ) ( n = 11 ) samples from indicated studies . Exact p values determined by Mann - Whitney .

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C, D. Log2 median-centered ROCK1 or ROCK2 RNA expression in normal (n=39) vs PDAC (n=39), normal (n=5) vs pancreatic adenocarcinoma (PAC) (n=12), or normal (n=6) vs pancreatic carcinoma (PC) (n=11) samples from indicated studies. Exact p values determined by Mann-Whitney test.


E . ROCK1 ROCK2 mRNA expression in human PDAC ( n = 146 ) from TCGA research network . Significance ( p value ) of slope deviation from 0 determined by Deming regression .

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E. ROCK1 and ROCK2 mRNA expression in human PDAC (n=146) from the TCGA research network. Significance (p value) of slope deviation from 0 determined by Deming regression.


F . Representative hematoxylin eosin ( H & amp ; amp ; E ) ROCK2 immunohistochemistry stained sections of normal mouse pancreas , acinar - to - metaplasia ( ADM ) , pancreatic intraepithelial neoplasia ( PanIN ) stages 1 - 3 , PDAC from indicated genotype mice . Scale bar = 50 µm .

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F. Representative hematoxylin and eosin (H&amp;E) and ROCK2 immunohistochemistry stained sections of normal mouse pancreas, acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1-3, and PDAC from indicated genotype mice. Scale bar = 50 µm.


G . of ROCK2 in epithelial of normal , ADM / PanIN1 ( KC ) , PanIN2 ( KPC ) , PanIN3 ( KPC ) PDAC ( KPC ) pancreatic tissue ( n = 5 mice per group ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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G. Quantification of ROCK2 staining in epithelial cells of normal, ADM/PanIN1 (KC), PanIN2 (KPC), PanIN3 (KPC) and PDAC (KPC) pancreatic tissue (n=5 mice per group). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


H . Survival of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; LSL - ROCK2 : ER ( RKPC ) mice without ( n = 21 ) or with conditional ROCK activation with tamoxifen citrate ( n = 19 ) . Survival p value determined by Log - Rank .

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H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.


A . H & amp ; amp ; E stained sections of invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPC in presence or absence of 10 µM H1152 ROCK inhibitor . Means ± ( n = 9 for untreated , n = 8 for H1152 ) , p value by unpaired t .

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

A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test.


B . Schematic representation of ROCK domains ( RBD , Rho binding domain ; PH , pleckstrin homology domain ; CR , cysteine - rich ) . Conditionally - activated human ROCK1 , human ROCK2 GFP control fusion ( EGFP , enhanced green ; hbER , estrogen receptor hormone binding domain ) were expressed in KPflC mouse PDAC blotted with anti - GFP antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally-activated human ROCK1, human ROCK2 and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody.


C . KPflC expressing GFP : ER , ROCK1 : ER or ROCK2 : ER fusion were treated for 24 h with EtOH vehicle or 1 µM 4HT in presence or absence of 1 µM or 10 µM H1152 . Immunoblotting shows endogenous ROCK1 ROCK2 , ER - fusions , phosphorylation of MLC2 ( T18S19 ) . Total MLC ( MRCL3 / MRLC2 / MYL9 ) glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) were blotted as loading controls .

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

C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.


D . H & amp ; amp ; E stained sections of invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPflC treated with 1 µM 4HT . Means ± ( n = 6 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison .

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D. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.


E . proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . F . of number at collagen matrix surface per 0 . 046 mm2 field . Means ± ( n = 30 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison . G . Ki67 positive percentages at surface within collagen matrix . Means ± ( n = 30 ; n = 12 for GFP : ER / Matrix ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison .

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E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=30), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.G. Ki67 positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n=30; n=12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.


H . Viable relative to starting numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on uncoated plastic surfaces . Means ± ( n = 3 ) . I . Viable relative to starting numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on collagen1 - coated surfaces . Means ± ( n = 3 ) .

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

H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).


A . Immunofluorescence of collagen matrix sections co - stained for collagen neo - epitope Col1 3 / 4C DAPI ( left panels ) . Second harmonic generation ( SHG ) of collagen within 3 - dimensional collagen matrices at 15 µm 45 µm below surface ( middle panels ) . Scale bar = 20 µm . Graphs indicate mean ± fibrillary collagen areas at 2 . 5 µm increments below / collagen interface ( n = 5 ) . SHG fibrillar collagen ( red ) ; ( green ) . B , C . Grey level correlation matrix ( GLCM ) texture of SHG correlation ( n = 6 ) contrast ( n = 5 ) . Mean ± at each .

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A. Immunofluorescence of collagen matrix sections co-stained for cleaved collagen neo-epitope Col1 3/4C and DAPI (left panels). Second harmonic generation (SHG) images of collagen fibers within 3-dimensional collagen matrices at 15 µm and 45 µm below the cell surface (middle panels). Scale bar = 20 µm. Graphs indicate the mean ± SEM fibrillary collagen areas at 2.5 µm increments below the cell/collagen interface (n=5). SHG fibrillar collagen (red); cells (green).B, C. Grey level correlation matrix (GLCM) texture analysis of SHG correlation (n=6) and contrast (n=5). Mean ± SEM at each distance.


D . Transmission electron microscopy of a collagen matrix section . Scale bar = 1 µm . Positions of magnified areas indicated by corresponding colored squares .

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D. Transmission electron microscopy of a collagen matrix section. Scale bar = 1 µm. Positions of magnified areas indicated by corresponding colored squares.


E . Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid . Yellow arrows indicate long collagen bundles , Red arrows indicate short collagen bundles . Scale bar = 0 . 5 µm .

E. Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid. Yellow arrows indicate long collagen bundles, Red arrows indicate short collagen bundles. Scale bar = 0.5 µm.


A . Volcano plots of sequencing data of genes that pass a threshold false discovery rate ( FDR ) < 5 % , with log10 fold - change ( FC ) in expression in 4HT treated ROCK1 : ER expressing relative to GFP : ER expressing ( left ) or 4HT treated ROCK2 : ER expressing relative to GFP : ER expressing ( right ) versus log10 adjusted p value ( n = 3 ) . ROCK1 : ER + 4HT vs GFP : ER + 4HT ; 3828 genes . ROCK2 : ER + 4HT vs GFP : ER + 4HT ; 4481 genes .

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A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes.


C . of 33 differentially expressed genes in MetaCoreTM process network adhesion : - matrix interaction . High relative expression in red low relative expression in blue .

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C. Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion: Cell-matrix interaction. High relative expression in red and low relative expression in blue.


D . MMP average reads . Mean ± ( n = 3 ) * adjusted p - value < 0 . 05 .

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D. MMP average RNA sequence reads. Mean ± SEM (n=3) * adjusted p-value <0.05.


E . Mmp10 mRNA relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± ( n = 4 ) , p value by unpaired t . Mmp10 mRNA relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison . F . Mmp13 mRNA relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) or 1 µM 4HT for 24 h ( left ) . Means ± ( n = 4 ) , p value by unpaired t . Mmp13 mRNA relative to Gapdh determined by qPCR following treatment with EtOH vehicle ( - ) , 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h ( right ) . Means ± ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


G . MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma . Scale bar = 100 µm ( left ) . Histoscores of MMP10 ( right ) in normal pancreas ( n = 5 ) , pancreas adenocarcinoma grade 1 ( n = 23 ) , grade 2 ( n = 27 ) grade 3 ( n = 13 ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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G. MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma. Scale bar = 100 µm (left). Histoscores of MMP10 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma grade 1 (n=23), grade 2 (n=27) and grade 3 (n=13). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


A . Confocal microscope of ROCK1 : ER - expressing co - stained for F - actin , MMP10 ( left ) or MMP13 ( right ) DAPI following treatment with vehicle or 1 µM 4HT for 24 h . Multiple z - planes were used to generate x - z y - z . Scale bar = 5 µm .

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A. Confocal microscope images of ROCK1:ER-expressing cells co-stained for F-actin, MMP10 (left) or MMP13 (right) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h. Multiple z-planes were used to generate x-z and y-z images. Scale bar = 5 µm.


B . Representative immunoblot of MMP13 MMP10 in conditioned media , as well as MMP10 GAPDH in lysates . were treated with vehicle ( - ) or 1 µM 4HT for 48 h .

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B. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (-) or 1 µM 4HT for 48 h.


C . Representative stained gel of ultracentrifuge - enriched microvesicle from GFP : ER or ROCK1 : ER - expressing - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h ( top ) . Absolute arbitrary unit values for total microvesicle in stained gels ( bottom ) . Means ± ( n = 4 ) , p value by paired t - .

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C. Representative stained gel of ultracentrifuge-enriched microvesicle protein from GFP:ER or ROCK1:ER-expressing cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM (n=4), p value by ratio paired t-test.


D . Transmission electron microscopy of ultracentrifuge - enriched microvesicles ( top center ) , immunogold labeling of MMP10 in ROCK1 : ER 4HT - treated microvesicles , indicated by red arrows ( top right ) .

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D. Transmission electron microscopy of ultracentrifuge-enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT-treated microvesicles, indicated by red arrows (top right).


D . Representative immunoblot of MMP10 caveolin in ultracentrifuge - enriched microvesicle from - conditioned media following treatment with vehicle ( - ) or 1 µM 4HT for 24 h as well as MMP10 , caveolin GAPDH in lysates ( top left ) . Absolute arbitrary unit values for MMP10 in western blots of ultracentrifugation - enriched microvesicle ( bottom left ) . Means ± ( n = 4 ) , p value by paired t . Ratios of MMP10 to caveolin ( bottom center ) or to total microvesicle ( bottom right ) . Means ± ( n = 4 ) , p value by paired t .

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D. Representative immunoblot of MMP10 and caveolin in ultracentrifuge-enriched microvesicle proteins from cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h as well as MMP10, caveolin and GAPDH in cell lysates (top left). Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation-enriched microvesicle proteins (bottom left). Means ± SEM (n=4), p value by ratio paired t test. Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM (n=4), p value by ratio paired t test.


E . Absolute arbitrary unit values for caveolin in western blots of ultracentrifugation - enriched microvesicle . Means ± ( n = 4 ) , p value by paired t . of caveolin to total microvesicle . Means ± ( n = 4 ) , p value by paired t . F . Ratios of MMP10 to GAPDH caveolin to GAPDH in western blots of whole lysates . Means ± ( n = 3 ) , p value by paired t .

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E. Absolute arbitrary unit values for caveolin levels in western blots of ultracentrifugation-enriched microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test. Ratio of caveolin to total microvesicle proteins. Means ± SEM (n=4), p value by ratio paired t test.F. Ratios of MMP10 to GAPDH and caveolin to GAPDH in western blots of whole cell lysates. Means ± SEM (n=3), p value by ratio paired t test.


A . Representative of treated with 1 µM 4HT in absence or presence of 10 µM H1152 for 18 h on Collagen1 - FITC ( Col1 - FITC ) . Co - for F - actin , MMP10 ( red ) DAPI ( blue ) . Scale bar = 10 µm . B . of collagen degradation by treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h . Means ± ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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A. Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1-FITC (Col1-FITC). Co-staining for F-actin, MMP10 (red) and DAPI (blue). Scale bar = 10 µm.B. Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


C . of collagen degradation by treated with 1 µM 4HT , 4HT + DMSO vehicle , 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h . Means ± ( n = 15 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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C. Quantification of collagen degradation by cells treated with 1 µM 4HT, 4HT + DMSO vehicle, 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


D . Immunofluorescence of collagen matrix sections co - stained for MMP10 DAPI . Scale bar = 20 µm .

D. Immunofluorescence of collagen matrix sections co-stained for MMP10 and DAPI. Scale bar = 20 µm.


E . Mmp13 immunohistochemistry stained sections of collagen matrices . Scale bar = 50 µm .

E. Mmp13 immunohistochemistry stained sections of collagen matrices. Scale bar = 50 µm.


A . H & amp ; amp ; E stained sections of invasion into collagen matrix after 8 days , in absence ( top ) or presence of 10 µM GM6001 ( bottom ) . Scale bar = 100 µm . B . Invasion index of KPflC . Means ± ( n = 4 ) , p value by unpaired t . C . of number at collagen matrix surface per 0 . 046 mm2 field . Means ± ( n = 20 ) , p value by unpaired t .

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A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 µM GM6001 (bottom). Scale bar = 100 µm.B. Invasion index of KPflC cells. Means ± SEM (n=4), p value by unpaired t test.C. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=20), p value by unpaired t test.


D . proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . E . Ki67 positive percentages at collagen matrix surface . Means ± ( n = 20 ; n = 19 for GFP : ER / vehicle , n = 18 for GFP / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison . F . Ki67 positive percentages in collagen matrix . Means ± ( n = 20 ; n = 8 for GFP : ER / vehicle , n = 10 for GFP : ER / GM6001 , n = 17 for ROCK1 : ER / GM6001 , n = 15 for ROCK2 : ER / GM6001 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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D. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.E. Ki67 positive cell percentages at the collagen matrix surface. Means ± SEM (n=20; n=19 for GFP:ER/vehicle, n=18 for GFP/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Ki67 positive cell percentages in collagen matrix. Means ± SEM (n=20; n=8 for GFP:ER/ vehicle, n=10 for GFP:ER/GM6001, n=17 for ROCK1:ER/GM6001, n=15 for ROCK2:ER/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


G . increase in viable numbers of plated on uncoated plastic surfaces treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001 . Means ± ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison . H . increase in viable numbers of plated on collagen1 ( Col1 ) coated surfaces treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001 . Means ± ( n = 3 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison .

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G. The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. The increase in viable cell numbers of cells plated on collagen1 (Col1) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.


A . Survival of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; ( KPC ) mice without ( n = 13 ) or with Fasudil treatment ( n = 13 ) . Survival p - value determined by Gehan - Breslow - Wilcoxon .

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A. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; (KPC) mice without (n=13) or with Fasudil treatment (n=13). Survival p-value determined by Gehan-Breslow-Wilcoxon test.


B . Representative of picrosirius red stained endpoint tumors from KPC mice . Scale bar = 1 mm . C . Picrosirius red positive ( top ) or average of positive ( bottom ) in pancreatic tumors from vehicle ( n = 8 ) Fasudil ( n = 7 ) treated mice . Box plots with exact p value by Mann - Whitney .

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B. Representative images of picrosirius red stained endpoint tumors from KPC mice. Scale bar = 1 mm.C. Picrosirius red percentage positive staining area (top) or average staining intensity of positive area (bottom) in pancreatic tumors from vehicle (n=8) and Fasudil (n=7) treated mice. Box plots with exact p value by Mann-Whitney test.


E . Ectopic expression of CG14044 in wing pouch ( GFP indicated by dotted ) induces caspase activation fragmentation ( TUNEL ) in larval disc , melanization in pupal wing ( arrow head ) a structure defect in adult wing . Data information : Scale bars , 100 μm in E ( caspase ) 20 μm in E ( TUNEL ) 500 μm in E ( pupae adult wing ) .

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E. Ectopic expression of CG14044 in the wing pouch (GFP area indicated by the dotted line) induces caspase activation and DNA fragmentation (TUNEL) in the larval disc, melanization in the pupal wing (arrow head) and a structure defect in the adult wing. Data information: Scale bars, 100 μm in E (cleaved caspase) and 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).


H . Deletion of BH3 motif suppresses Sayonara - induced caspase activation , which was detected by caspase sensor GC3Ai . I . of GC3Ai positive areas with Synr WT BH3 motif deletion mutant . Data information : Statistical significance was determined using one - way ANOVA with Dunnett ' s post hoc . Scale bars , 100 μm

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H. Deletion of the BH3 motif suppresses Sayonara-induced caspase activation, which was detected by the caspase sensor GC3Ai. I. Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant. Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in


A - D . Synr - induced wing structural defect is suppressed by knockdown of Debcl , Buffy or DCP1 . Data information : Scale bars , 500 μm in

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A-D. The Synr-induced wing structural defect is suppressed by knockdown of Debcl, Buffy or DCP1. Data information: Scale bars, 500 μm in A


F . Synr - induced caspase activation is suppressed by knockdown of Debcl . G . GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information : Scale bars , 50 μm in F

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F. Synr-induced caspase activation is suppressed by knockdown of Debcl. G. GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information: Scale bars, 50 μm in F and G


H - I . Lysates from HEK293T that express Flag - Debcl or Flag - Buffy were incubated with GST or GST - Synr bound to glutathione - Sepharose . Flag - Debcl or Flag - Buffy is pulled down with GST - Synr but not with GST .

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H-I. Lysates from HEK293T cells that express Flag-Debcl or Flag-Buffy were incubated with GST or GST-Synr bound to glutathione-Sepharose. Flag-Debcl or Flag-Buffy is pulled down with GST-Synr but not with GST.


J . Deletion of neither coiled coil domain nor BH3 motif suppresses binding Flag - Debcl to GST - Synr .

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J. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag-Debcl to GST-Synr.


O . Debcl can be photo - crosslinked with Synr at BPA that corresponds to Y124 F141 . BPA at Y340 was used as a negative control . Red arrowheads indicate covalently crosslinked products . P . Buffy is also photo - crosslinked with Synr at BPA that corresponds to Y124 F141 . Red arrowheads indicate covalently crosslinked products .

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O. Debcl can be photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. BPA at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products. P. Buffy is also photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.


R . HA - Debcl is detected after two series of immunoprecipitation . first immunoprecipitation was performed for Flag - Buffy , followed by Flag peptide - mediated elution secondary precipitation for Myc - Synr . HA - Debcl can be detected after this sequential immunoprecipitation .

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R. HA-Debcl is detected after two series of immunoprecipitation. The first immunoprecipitation was performed for Flag-Buffy, followed by Flag peptide-mediated elution and secondary precipitation for Myc-Synr. HA-Debcl can be detected after this sequential immunoprecipitation.


A . Ectopic expression of Synr induces autolysosome accumulation , which was detected by Lysotracker , in wing disc . B . Ectopic expression of Synr also induces autophagosome accumulation , which was detected by mCherry - atg8a . dotted square regions are magnified in right pictures . Data information : Scale bars , 100 μm in A H , 50 μm in

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A. Ectopic expression of Synr induces autolysosome accumulation, which was detected by Lysotracker, in the wing disc. B. Ectopic expression of Synr also induces autophagosome accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures. Data information: Scale bars, 100 μm in A and H, 50 μm in B


D - F . Knockdown of Atg2 or Atg8a suppresses wing structural defect that is induced by Synr . Data information : Scale bars , 500 μm in D .

D-F. Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr. Data information: Scale bars, 500 μm in D.


Synr - induced caspase activation is suppressed by inhibition of autophagy ( atg2 , atg8a RNAis ) in a similar manner to apoptosis inhibition ( Debcl , Dcp - 1 RNAis ) . Data information : Scale bars , 100 μm

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Synr-induced caspase activation is suppressed by inhibition of autophagy (atg2, atg8a RNAis) in a similar manner to apoptosis inhibition (Debcl, Dcp-1 RNAis). Data information: Scale bars, 100 μm in


A . Inhibition of Synr suppresses wing defect that is induced by p53 , a potent apoptosis inducer . B . Inhibition of Synr suppresses p53 - induced caspase activation . Data information : Statistical significance was determined using a two - unpaired t - ( B ) , Scale bar , 500 μm in

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A. Inhibition of Synr suppresses the wing defect that is induced by p53, a potent apoptosis inducer. B. Inhibition of Synr suppresses p53-induced caspase activation. Data information: Statistical significance was determined using a two-tailed unpaired t-test (B), Scale bar, 500 μm in A


D . Inhibition of Synr in midgut enterocytes enhances resistance to starvation . E . synr mutant also exhibits enhanced resistance to starvation . Data information : Statistical significance was determined using a log - rank ( Mantel - Cox ) ( D - E ) .

D. Inhibition of Synr in the midgut enterocytes enhances resistance to starvation. E. The synr mutant also exhibits enhanced resistance to starvation. Data information: Statistical significance was determined using a log-rank (Mantel-Cox) test (D-E).


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Survival ( n = 5 ) in infected mice were monitored daily Data information : Log - rank was

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Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in infected mice were monitored daily Data information: Log-rank test was used


Four to six - week - old C57BL / 6 mice were injected i . p . with 104 - 107 PFU of WNV - poly ( A ) , 104 PFU of WT WNV or PBS ( negative control ) in a volume of 200 μL . Histological of mouse brain . Scale bars represent 100 μm 50 μm for left right panels , respectively . Severe vacuolization ( black arrows ) inflammation ( yellow arrow ) in brain small vessels as well as degenerative changes in neurons with rarefaction ( blue arrow ) necrosis with compaction ( red arrow ) were indicated . Scale bars represent 100 μm 50 μm for left right panels ,

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Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Histological analysis of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with cytoplasmic rarefaction (blue arrow) and necrosis with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectively


E Viral load replication kinetics in different tissues of C57BL / 6 mice . Six - week - old C57BL / 6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV - poly ( A ) in left rear footpad , tissues were harvested from two mice at indicated times post - inoculation . Viral loads in brain , spleen , left lymph node , right lymph node liver were measured by plaque expressed as PFU per gram of tissue . n = 3 , values are mean ± sd , dotted corresponds to limit of detection ( LOD ) .

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E Viral load and replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the left rear footpad, and tissues were harvested from two mice at the indicated times post-inoculation. Viral loads in brain, spleen, left lymph node, right lymph node and liver were measured by plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).


B - C Total anti - WNV IgG antibody ( B ) neutralizing antibody titers ( C ) in mouse serum at indicated days post immunization . ( For B C , values represent mean ± sd in each group horizontal dotted represents limit of detection ) Data information : For statistical , one - way ANOVA were used * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection) Data information: For statistical analysis, one-way ANOVA analysis were used *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


D - E Survival ( D ) - changes were monitored daily after challenge . ( values represent mean ± sd in each group ) ( E ) . Data information : For statistical , two - way ANOVA were used Log - rank was performed * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

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D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). Data information: For statistical analysis, two-way ANOVA analysis were used Log-rank test was performed *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


G - H T - responses in vaccinated mice were measured through ELISpot . Two groups of C57BL / 6 mice ( n = 4 / group ) were i . p . inoculated with 107 of WNV - poly ( A ) or equal volume of PBS . ELISpot specific for IFN - γ in splenocytes was performed at 7 days post - immunization . Representative were shown in ( G ) numbers of antibody secreting ( ASCs ) were counted ( values represent mean ± sd in each group , each symbol represents a mouse ) ( H ) . Data information : Mann - Whitney was used for H . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not

O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O 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 T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant


C Comparison of growth kinetics between WT WNV WNV - poly ( A ) in MEF . MEF were infected with either WT WNV or WNV - poly ( A ) at an MOI of 0 . 01 . Values indicate mean ± sd from two replicates of one representative experiment . Data information : Three independent assays were performed for above experiments with similar results data of one representative data was shown . Scale bars represent 50 μm .

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C Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment. Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


D Survival rate of IFNAR - / - mice infected with different dosages of WNV - poly ( A ) ( n = 5 ) . Data information : Three independent assays were performed for above experiments with similar results data of one representative data was shown . Scale bars represent 50 μm .

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D Survival rate of IFNAR-/- mice infected with different dosages of WNV-poly(A) (n=5). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


E changes of MEF infected with WT WNV WNV - poly ( A ) . Data information : Three independent assays were performed for above experiments with similar results data of one representative data was shown . Scale bars represent 50 μm .

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E Cell morphology changes of MEF cells infected with WT WNV and WNV-poly(A). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.


Virulence efficacy of passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( E ) Survival of mice infected with 107 PFU of WNV - poly ( A ) - P50 . Data information : For statistical , one - way ANOVA Log - rank were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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

Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


Virulence efficacy of passaged WNV - poly ( A ) ( WNV - poly ( A ) - P50 ) in mice ( n = 5 ) . ( F ) Antibody titers of mice infected with WNV - poly ( A ) - P50 at different days post immunization . ( For values represent mean ± sd in each group ( n = 5 ) horizontal dotted represents limit of detection ) . Data information : For statistical , Mann - Whitney were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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

Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


( I ) Survival of mice after WT WNV challenge . Data information : For statistical , one - way ANOVA Log - rank were used * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns represents not significant .

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(I) Survival of mice after WT WNV challenge. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.


( A ) Immunoblots for LC3 using whole lysates of Huh7 before after treatment with iron chelators ( DFP , DFX or DFO ) . β - actin was used as loading control . Bafilomycin ( - ) : without Bafilomycin , Bafilomycin ( + ) : with 100 mM of Bafilomycin .

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(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin.


( B ) Dual excitation ( 550 / 488 nm ) of mt - mKeima transfected - Huh7 shows red indicating mitophagy with DFP treatment at 24 h green without DFP treatment . Scale bar : 10 μm .

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(B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm.


( C ) high signal were quantified by flow cytometry with dual excitation of mt - mKeima at 633 / 488 nm for untreated ( left panel ) DFP - treated ( 24 h , right panel ) Huh7 .

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(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.


( D ) Untreated Huh7 or those treated with DFP ( 10 μM ) , DFX ( 10 μM ) or DFO ( 10 μM ) were quantified for mitophagy using mt - mKeima transfection FACS ( n = 4 , biological replicates ) . A high signal ( 633 / 488 ) / indicates proportion of undergoing mitophagy . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * * : P < 0 . 01 .

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(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.


( E ) Electron microscopic of untreated ( left panel ) DFP - treated ( right panel ) Huh7 . Arrows indicate mitophagosome - like structures . Scale bar : 500 nm . number of mitophagosome - like structures / 100 μm2 was compared for four randomly selected areas ( technical replicates ) between treated with DFP ( 1 . 0 mM ) those without DFP . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 .

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(E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.


( F ) of mitophagy using mt - mKeima transfection FACS for Huh7 treated with DFP ( 0 , 0 . 01 . 0 . 5 or 1 . 0 mM as indicated ) in absence or presence of bafilomycin ( n = 4 , biological replicates ) . central horizontal bar error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

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(F) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( G ) Immunoblots for Parkin , PINK1 , phosphorylated p - 62 ( p - p62 ) , HSP60 , LC3 , p62 / SQSTM , Atg5 using mitochondrial fraction whole lysates of Huh7 . HSP60 β - actin were used as loading controls for mitochondria whole lysates , respectively .

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(G) Immunoblots for Parkin, PINK1, phosphorylated p-62 (p-p62), HSP60, LC3, p62/SQSTM, and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells. HSP60 and β-actin were used as loading controls for mitochondria and whole cell lysates, respectively.


( A ) mitochondrial iron content was measured by a ferrozine - for Huh7 with DFP , DFX or DFO treatment ( 0 . 01 mM ) for 24 h ( n = 5 , biological replicates ) . iron content was measured by subtracting iron content of mitochondrial fraction from iron content of whole lysate . central horizontal bar error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

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(A) Cytoplasmic and mitochondrial iron content was measured by a ferrozine-based assay for Huh7 cells with DFP, DFX or DFO treatment (0.01 mM) for 24 h (n=5, biological replicates). The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate. The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( B ) mitochondrial iron content was measured for Huh7 with DFP treatment ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) for 24 h . ( n = 5 , biological replicates ) central horizontal bar error bards indicate mean ± SD . * : P < 0 . 05 , * * : P < 0 . 01 .

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(B) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated) for 24 h. (n=5, biological replicates) The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.


( C ) Immunoblots ( left panel ) for mitochondrial ferritin ( FTMT ) , frataxin , mitoferrin , heat shock 60 ( HSP60 ) , ferritin heavy chain ( FTH1 ) transferrin receptor ( TfR ) β - actin using mitochondrial fraction or whole lysate of Huh7 following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM as indicated ) . HSP60 β - actin were used as loading controls for mitochondrial whole lysates , respectively . expression level of FTMT was normalized to HSP60 ( n = 4 , biological replicates , right panel ) . central horizontal bar error bards indicate mean ± SD . * : P < 0 . 05 .

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(C) Immunoblots (left panel) for mitochondrial ferritin (FTMT), frataxin, mitoferrin, heat shock protein 60 (HSP60), ferritin heavy chain (FTH1) and transferrin receptor (TfR) and β-actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated). HSP60 and β-actin were used as loading controls for mitochondrial and whole cell lysates, respectively. The expression level of FTMT was normalized to HSP60 (n=4, biological replicates, right panel). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05.


( D ) mRNA expression of FTMT in Huh7 was quantified by real - time RT - PCR before after DFP ( 1 . 0 mM ) treatment ( n = 4 , biological replicates ) . expression of FTMT mRNA level was normalized to GAPDH mRNA . central horizontal bar error bards indicate mean ± SD . Two sample t was used for statistical . * * : P < 0 . 01 .

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(D) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before and after DFP (1.0 mM) treatment (n=4, biological replicates). The expression of FTMT mRNA level was normalized to GAPDH mRNA. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.


( E ) mRNA expression of FTMT in Huh7 was quantified by real - time RT - PCR before DFP ( 1 . 0 mM ) treatment 3 , 6 , 12 , 24 h after DFP treatment ( n = 5 , biological replicates ) . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * * : P < 0 . 01 , * * * : P < 0 . 001 .

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(E) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before DFP (1.0 mM) treatment and 3, 6, 12, and 24 h after DFP treatment (n=5, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01, ***: P<0.001.


( F ) - binding activity of six transcription factors ( specific 1 [ SP1 ] , cAMP response element binding [ CREB ] , Ying yang 1 [ YY1 ] , GATA2 , forkhead box A1 [ FoxA1 ] CCAAT - enhancer - binding β [ C / EBPβ ] ) to FTMT promoter region was compared by immunoprecipitation before after DFP ( 1 . 0 mM ) treatment in Huh7 ( n = 4 , biological replicates ) . central horizontal bar error bards indicate mean ± SD . Two sample t was used for statistical . * : P < 0 . 05 .

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(F) DNA-binding activity of six transcription factors (specific protein 1 [SP1], cAMP response element binding protein [CREB], Ying yang 1 [YY1], GATA2, forkhead box protein A1 [FoxA1] and CCAAT-enhancer-binding protein β [ C/EBPβ]) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP (1.0 mM) treatment in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. *: P<0.05.


( G ) - binding activity of SP1 to FTMT promoter region was assayed by immunoprecipitation following treatment with DFP ( 0 , 0 . 01 , 0 . 1 , 0 . 5 or 1 . 0 mM ) as indicated in Huh7 ( n = 4 , biological replicates ) . central horizontal bar error bards indicate mean ± SD . Tukey ' s honestly significant was used for statistical . * : P < 0 . 05 , * * : P < 0 . 01 .

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(G) The DNA-binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM) as indicated in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.