( B ) Validation of the neutralization method was carried out with cell HEK - 293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein . The supernatants were mixed with sera from donor # 66 at the indicated dilutions or from a pre - COVID sample as a control before addition onto HEK - 293T - ACE2 + cell cultures . No serum , refers to a control containing no human sera . Data represent the mean ± SD of triplicates .

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

(B) Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum, refers to a control containing no human sera. Data represent the mean±SD of triplicates.


( C ) The presence of neutralizing antibodies in sera from donors # 46 and # 48 , found negative by ELISA and positive by flow cytometry ( Fig . 3C ) was demonstrated as in panel B . Data represent the mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample ) .

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

(C) The presence of neutralizing antibodies in sera from donors #46 and #48, found negative by ELISA and positive by flow cytometry (Fig. 3C) was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( D ) Bar plot showing the S / EGFR MFI ratio determined by flow cytometry analysis with Jurkat - S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS - CoV - 2 at the Ramón y Cajal Hospital of Madrid . A negative result is considered for a ratio lower than 0 . 5 . Two clear cases of sera positive for anti - S IgG1 are indicated ( RyC52 and RyC65 ) . A borderline sample just above the threshold line ( RyC58 ) is also indicated . Data show the mean ± s . e . m . All datapoints are shown .

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

(D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown.


( F ) Plot of the S / EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S / EGFR and shape ( see Materials and Methods and Fig . EV1 ) . The green line indicates the threshold point ( 0 . 024 ) for the Score above which a serum is considered positive .

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 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

(F) Plot of the S/EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig. EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive.


( G ) The presence of neutralizing antibodies in sera from donors RyC46 , RyC52 , RyC56 and RyC58 diluted 1 / 15 was demonstrated as in panel B . Data represent the mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Unpaired two - tailed t - test comparing each serum to the No serum sample )

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

(G) The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56 and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Unpaired two-tailed t-test comparing each serum to the No serum sample)


( A ) A total of 52 serum samples from Hospital Universitario La Princesa ( HUP ) and 52 samples of pre - COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S / EGFR - based Score . Of the 52 HUP samples , a total of 40 were from donors testing positive by PCR and by an ELISA test , 6 were from donors testing negative by PCR but positive by ELISA , 1 from an individual testing negative by ELISA and positive by PCR , and 5 were not tested by PCR . Box and whiskers are shown to represent the minimum and maximum values as well as the median . All datapoints are shown . The broken line indicates the threshold for a Score value of 0 . 024 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A total of 52 serum samples from Hospital Universitario La Princesa (HUP) and 52 samples of pre-COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S/EGFR-based Score. Of the 52 HUP samples, a total of 40 were from donors testing positive by PCR and by an ELISA test, 6 were from donors testing negative by PCR but positive by ELISA, 1 from an individual testing negative by ELISA and positive by PCR, and 5 were not tested by PCR. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown. The broken line indicates the threshold for a Score value of 0.024.


C ) Four HUP serum samples giving discrepant results by flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus ( Fig . 4A ) . Data represent the mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ; ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample )

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

C) Four HUP serum samples giving discrepant results by flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus (Fig. 4A). Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ****, p<0.00005; (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample)


( A ) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat - S cells at the indicated dilutions . Flow cytometry data are shown as the S / EGFR MFI ratio . Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer .

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

(A) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat-S cells at the indicated dilutions. Flow cytometry data are shown as the S/EGFR MFI ratio. Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer.


( B ) A neutralization assay with S protein - pseudotyped lentivirus was carried out to resolve the discrepancies . Data represent the mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0005 ; * * * * * , p < 0 . 000005 ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample ) .

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

(B) A neutralization assay with S protein-pseudotyped lentivirus was carried out to resolve the discrepancies. Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ***, p<0.0005; *****, p<0.000005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( C ) Classification of the CBMSO cohort according to the Score generated from S / EGFR data . The Broken line indicates the 0 . 024 threshold above which samples are considered positive .

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

(C) Classification of the CBMSO cohort according to the Score generated from S/EGFR data. The Broken line indicates the 0.024 threshold above which samples are considered positive.


( D ) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel ( top - down view ) ; wing disc resting on a stretching device ( anchor ) and 10 min after stretch ; M = microchannel .

O O O O O O O O O O O O O O O O O O O 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) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel.


( E ) zipper RNAi clones ( zip iR , in red ) in Arm : : GFP expressing anchored and stretched third instar wing disc ; lower panel shows color coded cell aspect ratio in anchored and stretched discs ( bright colors indicate high elongation ) ; zip RNAi clone is outlined in white .

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

(E) zipper RNAi clones (zip iR, in red) in Arm::GFP expressing anchored and stretched third instar wing disc; lower panel shows color coded cell aspect ratio in anchored and stretched discs (bright colors indicate high elongation); zip RNAi clone is outlined in white.


( F ) Box plot showing distribution of cell aspect ratio for control ( wild - type ) and zipper RNAi cells in anchored and stretched ( 20 min ) wing disc ; median represented by horizontal line , 75th and 25th centiles are represented by top and bottom of the boxes respectively ; n = 8 wing discs .

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

(F) Box plot showing distribution of cell aspect ratio for control (wild-type) and zipper RNAi cells in anchored and stretched (20 min) wing disc; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 wing discs.


( G ) Color - coded aspect ratio in control and zip RNAi cells ( zip RNAi clone outlined in white ) subjected to anchoring , stretching ( 20 min ) and relaxation ( 10 min , post stretch ) .

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

(G) Color-coded aspect ratio in control and zip RNAi cells (zip RNAi clone outlined in white) subjected to anchoring, stretching (20 min) and relaxation (10 min, post stretch).


( H ) Box plot showing distribution of cell aspect ratio in anchored , stretched ( 20 min ) and relaxed ( 10 min ) wing disc for control ( wild - type ) and zipper RNAi cells ; median represented by horizontal line ; 75th and 25th centiles are represented by top and bottom of the boxes respectively , n = 3 wing discs .

O O O O O O O O O O O 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

(H) Box plot showing distribution of cell aspect ratio in anchored, stretched (20 min) and relaxed (10 min) wing disc for control (wild-type) and zipper RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 wing discs.


( I ) zip - RNAi clone in Arm : : GFP wing disc at the beginning ( 5 min ) and at the end ( 40 min ) of stretching experiment . Yellow and red arrows indicate the control and zip RNAi cells respectively .

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

(I) zip-RNAi clone in Arm::GFP wing disc at the beginning (5 min) and at the end (40 min) of stretching experiment. Yellow and red arrows indicate the control and zip RNAi cells respectively.


( J ) Change of zip - RNAi aspect ratio ( a . r . ) relative to change in control cells aspect ratio ( a . r . ) in the course of stretch , blue line shows fitted regression ; dotted line represents 95 % confidence bounds of the best fit line ; n = 3 wing discs .

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

(J) Change of zip-RNAi aspect ratio (a.r.) relative to change in control cells aspect ratio (a.r.) in the course of stretch, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 wing discs.


( A ) Wing disc expressing E - cad : : GFP and Sqh : : mCherry prior to stretch ( anchor ) and after stretch ( 15 min ) . ( A " ) , Insets of discs shown in ( A ) . ( B ) Schematics demonstrating change in Sqh concentration before and after stretch on vertical ( V ) and horizontal ( H )

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

(A) Wing disc expressing E-cad::GFP and Sqh::mCherry prior to stretch (anchor) and after stretch (15 min).
(A"), Insets of discs shown in (A).
(B) Schematics demonstrating change in Sqh concentration before and after stretch on vertical (V) and horizontal (H) junctions


( C ) Quantification of mean fluorescent intensity of E - cad : : GFP and Sqh : : mCherry on horizontal ( H ) junctions relative to the intensity on vertical ( V ) junctions ( referred to as " polarity " ) prior to and after 15 min stretch . The detailed rationale of intensity determination is described in Fig . S3 and Experimental Procedures .

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

(C) Quantification of mean fluorescent intensity of E-cad::GFP and Sqh::mCherry on horizontal (H) junctions relative to the intensity on vertical (V) junctions (referred to as "polarity") prior to and after 15 min stretch. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.


( D - E ) , Quantification of E - cad : : GFP ( D ) and Sqh : : mCherry ( E ) concentration ( i . e . mean intensity per junctional unit area ) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched ( anchored ) vertical junctions .

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

(D-E), Quantification of E-cad::GFP (D) and Sqh::mCherry (E) concentration (i.e. mean intensity per junctional unit area) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched (anchored) vertical junctions.


( F ) Quantification of total amount of Sqh : : mCherry and E - cad : : GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs .

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

(F) Quantification of total amount of Sqh::mCherry and E-cad::GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs.


( G ) Tissue expressing E - cad : : GFP and Sqh : : mCherry subjected to increasing levels of stretch .

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

(G) Tissue expressing E-cad::GFP and Sqh::mCherry subjected to increasing levels of stretch.


( H ) Quantification of Sqh : : mCherry polarity ( as in c ) of experiment described in ( G ) . The level of stretch is measured as a change in tissue strain ( deformation relative to unstretched tissue ) .

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

(H) Quantification of Sqh::mCherry polarity (as in c) of experiment described in (G). The level of stretch is measured as a change in tissue strain (deformation relative to unstretched tissue).


( I - K ) Sqh : : mCherry polarity emergence as a function of cell aspect ratio change ( I ) , stress ( J ) , stress applied on average cell area of 10 μm2 ( K ) . Yellow asterisks indicate equivalent regions in the tissue . N = 8 wing discs ( A - F ) , N = 3 wing discs ( G - K ) ;

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

(I-K) Sqh::mCherry polarity emergence as a function of cell aspect ratio change (I), stress (J), stress applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the tissue. N=8 wing discs (A- F), N=3 wing discs (G-K);


( A ) Short ( 15 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs .

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

(A) Short (15 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( B ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( A ) ; schematics below the graph illustrates quantified changes in cell aspect ratio ( yellow ) and Sqh polarity ( red ) . Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc .

O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (A); schematics below the graph illustrates quantified changes in cell aspect ratio (yellow) and Sqh polarity (red). Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc.


( C ) Long ( 200 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs .

(C) Long (200 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( D ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( C ) ; schematics below the graph illustrates quantified changes in aspect ratio ( yellow ) and Sqh polarity ( red ) .

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

(D) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (C); schematics below the graph illustrates quantified changes in aspect ratio (yellow) and Sqh polarity (red).


( A ) Overview of the E - cad : : GFP disc prior to ( anchor ) and after compression .

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

(A) Overview of the E-cad::GFP disc prior to (anchor) and after compression.


( B ) E - cad : : GFP and Sqh : : mCherry localization in anchored and compressed wing disc .

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

(B) E-cad::GFP and Sqh::mCherry localization in anchored and compressed wing disc.


( C ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) stretching and short ( < 20 min ) compression . Plot shows increase in distance between recoiling short axis [ μm ] of the fitted ellipse ( see Fig . S5A for details ) against time [ sec . ] ; data is plotted as mean ± S . E . M , n = 7 - 10 discs / condition .

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

(C) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) stretching and short (<20 min) compression. Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 discs/condition.


( D ) Activated Myosin ( anti - phosphorylated - Sqh ; in red ) and anti - E - cad ( green ) staining in compressed discs . ( D " ) Close up of compression experiment described in ( D ) ; yellow arrow points towards an example of polarized phosphorylated - Sqh cable in compressed discs .

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

(D) Activated Myosin (anti-phosphorylated-Sqh; in red) and anti-E-cad (green) staining in compressed discs. (D") Close up of compression experiment described in (D); yellow arrow points towards an example of polarized phosphorylated-Sqh cable in compressed discs.


( A ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) and long ( > 120 min ) stretching and stretch retraction ( tissue forced back to the original , pre - stretch shape .

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

(A) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) and long (>120 min) stretching and stretch retraction (tissue forced back to the original, pre-stretch shape.


( C ) An overlay of a junction prior to ( red ) and 15 . 44 sec . after ablation ( green ) .

O 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) An overlay of a junction prior to (red) and 15.44 sec. after ablation (green).


( E ) Laser cuts ( perpendicular or parallel to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring or short stretching ( < 30 min ) . .

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

(E) Laser cuts (perpendicular or parallel to the line of stretch) across circa 10 cells in E-cad::GFP expressing wing disc tissue subjected to anchoring or short stretching (< 30 min)..


( H ) An overlay of anchored and stretched discs prior to ablation ( red ) and 15 . 44 seconds post ablation ( green ) . Inset zooms in on the area next to ablation site ( marked with yellow asterisk ) .

O O O O O O O O O O O O O O O O O O O O O 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) An overlay of anchored and stretched discs prior to ablation (red) and 15.44 seconds post ablation (green). Inset zooms in on the area next to ablation site (marked with yellow asterisk).


( A ) RokK116a : : Venus localization in anchored , stretched , relaxed third instar imaginal discs .

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

(A) RokK116a::Venus localization in anchored, stretched, relaxed third instar imaginal discs.


discs . ( B ) Drok2 mitotic clones ( RFP - ) in Sqh : : GFP third instar imaginal disc . Inset demonstrates Sqh : : GFP polarity inside Drok2 clone .

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

discs.
(B) Drok2 mitotic clones (RFP-) in Sqh::GFP third instar imaginal disc. Inset demonstrates Sqh::GFP polarity inside Drok2 clone.


( C ) Sqh : : GFP , enGal4 > UAS - rok RNAi , UAS - Act5CRFP discs subjected to stretching ; inset demonstrates Sqh : : GFP polarity in rok RNAi cells ( marked by red .

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

(C) Sqh::GFP, enGal4>UAS-rok RNAi, UAS-Act5CRFP discs subjected to stretching; inset demonstrates Sqh::GFP polarity in rok RNAi cells (marked by red.


( D ) zip RNAi clones ( labeled as zip iR , indicated in red ) in stretched third instar RokK116a : : Venus discs .

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

(D) zip RNAi clones (labeled as zip iR, indicated in red) in stretched third instar RokK116a::Venus discs.


( A ) Discs expressing Sqh : : GFP and enGal4 > UAS - dia RNAi , UAS - Act5c - RFP that were stretched ; UAS - Act5C : : RFP marks dia iR ( posterior side ) in red . ( A " ) Insets comparing Sqh : : GFP polarity in stretchedenGal4 > dia iR , UAS - Act5c - RFP discs ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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

(A) Discs expressing Sqh::GFP and enGal4>UAS-dia RNAi, UAS-Act5c-RFP that were stretched; UAS-Act5C::RFP marks dia iR (posterior side) in red. (A") Insets comparing Sqh::GFP polarity in stretchedenGal4>dia iR, UAS- Act5c-RFP discs; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( B ) Tissue marked with E - cad : : GFP and Sqh : : mCherry , expressing dia - RNAi ( dia iR ) with hhGal4 driver and subjected to stretching . Dotted line indicates anterior - posterior ( A - P ) compartment boundary . ( B " ) Inset demonstrates lack of Sqh : : GFP polarity in dia - RNAi cells .

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

(B) Tissue marked with E-cad::GFP and Sqh::mCherry, expressing dia-RNAi (dia iR) with hhGal4 driver and subjected to stretching. Dotted line indicates anterior-posterior (A-P) compartment boundary. (B") Inset demonstrates lack of Sqh::GFP polarity in dia-RNAi cells.


( C - D ) , Quantification of E - cad : : GFP polarity ( C ) and Sqh : : mCherry polarity ( D ) in experiment described in ( B ) ; polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions ( see Experimental Procedures for details ) ; n = 4 wing discs .

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

(C-D), Quantification of E-cad::GFP polarity (C) and Sqh::mCherry polarity (D) in experiment described in (B); polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions (see Experimental Procedures for details); n=4 wing discs.


( E ) Stretched discs expressing Sqh : : GFP and enGal4 > UAS - cofilin ( upper panel ) or UAS - AIP1 ( lower panel ) RNAi and UAS - Act5c - RFP ( marks posterior side in red ) . ( E " ) , Insets comparing Sqh : : GFP polarity in stretched discs described in ( E ) ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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

(E) Stretched discs expressing Sqh::GFP and enGal4>UAS-cofilin (upper panel) or UAS-AIP1 (lower panel) RNAi and UAS-Act5c-RFP (marks posterior side in red).
(E"), Insets comparing Sqh::GFP polarity in stretched discs described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( F ) Sqh : : GFP polarity in enGal4 > dia iR , UAS - Act5C : : RFP discs labeled with Phalloidin - 647 ; Inset ( Phall - 647 ) zooms in on cell shape in anterior and posterior disc compartments .

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

(F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments.


( G ) Discs expressing nubGal4 and Cd8 : : GFP ( control ) or dia iR ( KK ) labeled with DAPI ; ellipse is fitted into the pouch region ( red dotted line ) and aspect ratio determined as long ellipse axis ( b ) divided by short ellipse axis ( a ) ( white lines ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).


( H ) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in ( G ) , n = 19 ( ctrl ) and 16 ( dia iR ) wing pouches ; horizontal line indicates median .

O O O O O O 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

(H) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in (G), n=19 (ctrl) and 16 (dia iR) wing pouches; horizontal line indicates median.


( C ) Co - immunoprecipitation using BmN4 cells transfected with the Flag - tagged DEAD - box helicases identified by shotgun proteomics . The Flag - tagged proteins and Ago3 were detected by western blotting . Full - length Flag - tagged proteins are indicated with red asterisks . h . c . : heavy chain .

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

(C) Co-immunoprecipitation using BmN4 cells transfected with the Flag-tagged DEAD-box helicases identified by shotgun proteomics. The Flag-tagged proteins and Ago3 were detected by western blotting. Full-length Flag-tagged proteins are indicated with red asterisks. h.c.: heavy chain.


( F , G ) Analysis of Siwi - piRNA and Ago3 - piRNA levels in Siwi and Ago3 complexes with DDX43 , respectively . The WB ( western blotting ) and NB ( northern blotting ) signals represent the PIWI protein and piRNA levels , respectively ( see also Figs EV1A and EV1B ) . The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs .

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

(F, G) Analysis of Siwi-piRNA and Ago3-piRNA levels in Siwi and Ago3 complexes with DDX43, respectively. The WB (western blotting) and NB (northern blotting) signals represent the PIWI protein and piRNA levels, respectively (see also Figs EV1A and EV1B). The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs.


( B ) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3 .

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

(B) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3.


( C , D ) Immunoprecipitation of endogenous Siwi , DDX43 , and Vret bound with Ago3 from DDX43 - depleted ( C ) or Vret - depleted ( D ) cells . The factors were detected by western blotting . Ago3 interacts with Vret and DDX43 independently of the other .

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

(C, D) Immunoprecipitation of endogenous Siwi, DDX43, and Vret bound with Ago3 from DDX43-depleted (C) or Vret-depleted (D) cells. The factors were detected by western blotting. Ago3 interacts with Vret and DDX43 independently of the other.


( D ) RNA unwinding assays upon Ago3 - piRISC - dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage 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 but not Vasa released the cleaved target RNA from Ago3 - piRISC .

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

(D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage 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 but not Vasa released the cleaved target RNA from Ago3-piRISC.


( 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 .

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

(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 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 .

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

(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 ) 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 " .

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

(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 images of pulmonary fibrosis and quantification .

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


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

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

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


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

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


( 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 ) .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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 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

(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 and PF - PH patients .

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

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


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

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


( 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 .

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

(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 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 .

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

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

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

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


( C ) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and 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 protein ( PIP ) mRNA ( D ) normalized to GAPDH in PF and PF - PH patients .

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

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


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

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

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


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

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

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


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 .

O O O O 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

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 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 .

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

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 images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation .

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


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

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

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


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

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

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


( H ) Masson ' s trichrome representative images and quantification of Ashcroft score in the 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 ) and collagen III ( K ) mRNA normalized to GAPDH .

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

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


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

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


( 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 .

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

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

O O O O O O O O B-MEASURED_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 O O O O

(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 protein ( C ) normalized to GAPDH .

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


( 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 )

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

(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)


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

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


( 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 .

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

(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 protein ( B ) normalized to GAPDH .

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


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

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

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

O O O 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

(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 ) and Collagen III ( H ) normalized to GAPDH .

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

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


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

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

(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 images and quantification of the vascular remodeling between non - fibrotic and fibrotic areas of the lung .

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

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


( 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 .

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

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

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

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

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

(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 protein ( E ) normalized to GAPDH .

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


( F ) Representative images and quantification of PIP immunohistochemistry .

(F) Representative images and quantification of PIP immunohistochemistry.


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

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

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 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 -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 proteins bound specifically to H2AQ105me . Members of the FACT complex ( SPT16 / POB3 ) are highlighted in red .

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

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 and detected using streptavidine - HRP .

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

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


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 .

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-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

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

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

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 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 .

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

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 ) 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 ) .

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

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 .

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


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

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 proteins .

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 the mean between membranes and the size of the outline the standard deviation .

O O O O O O 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. 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 images in Appendix figure

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

A-C. 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 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 .

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

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 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 .

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

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 test with Bonferroni correction ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 The original data was taken from Xiong et al and included three biological replicates .

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

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 test with Bonferroni correction )

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

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 proteins from Glial cytoplasmic inclusions ( α - synuclein aggregates ) . ( statistics : hypergeometric test with Bonferroni correction )

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

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

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 images of colocalization of protein of interest with Aβ aggregates . ( Contrast of images were enhanced to 0 . 1 % saturated pixels using Fiji ) . Scale bars :

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

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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 ) .

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

(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 the p19ink4d ER8 , and the rarβ DR5 retinoic acid response element , as a control .

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

(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 ) 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 .

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

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

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

(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 ) 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 .

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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 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 .

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

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 B-MEASURED_VAR 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 cells . Extracts of U937 cells were probed for p27KIP1 and CDK4 , and immunoprecipitated with an anti ‐ CDK antibody and probed for p27KIP1 co ‐ immunoprecipitation .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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 RNA and protein in SCC25 cells . SiRNAs were directed against p19ink4d , scrambled p19ink4d ( Scr . ) or cyclophilin ( cyc . ) controls .

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

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

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

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


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 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 ) .

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 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 B-CONTROLLED_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 B-CONTROLLED_VAR 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 . Analysis of lysosomal fusion ( autophagosomes ) in SCC25 cells treated as in ( E ) , stained for incorporation of lysotracker red . Nuclei were counterstained with Hoechst ( blue ) .

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

(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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O 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. 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 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 .

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

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 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 .

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

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 and eosin ( H & amp ; 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 .

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

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 . 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 .

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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O 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. 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 .

O O O O O O O O O O O O O O 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. 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 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 .

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

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 . 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 .

O O O O O O O O O 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. 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 RNA sequence reads . Mean ± SEM ( n = 3 ) * adjusted p - value < 0 . 05 .

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

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


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 .

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 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 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 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

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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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

O O O O O B-MEASURED_VAR 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 O O O

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 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 .

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

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 ) , and immunogold labeling of MMP10 in ROCK1 : ER 4HT - treated microvesicles , indicated by red arrows ( top right ) .

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

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).