C . Transwell migration assay was performed using cells in ( A ) ( n = 8 ) . We analyzed 5 random fields for each sample . Error bars indicate the S . E . M . ( * * p < 0 . 01 , paired Student ' s t - test ) .

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C. Transwell migration assay was performed using cells in (A) (n=8). We analyzed 5 random fields for each sample. Error bars indicate the S.E.M. (**p< 0.01, paired Student's t-test).


D . Representative images of soft agar assay results using cells in ( A ) . Scale bar , 1 mm .

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D. Representative images of soft agar assay results using cells in (A). Scale bar, 1 mm.


E . Soft agar assay was performed using cells in ( A ) ( n = 8 ) . Error bars indicate the S . E . M . ( * * p < 0 . 01 , * * * p < 0 . 001 , paired Student ' s t - test ) .

E. Soft agar assay was performed using cells in (A) (n=8). Error bars indicate the S.E.M. (**p < 0.01, ***p < 0.001, paired Student's t-test).


B , C . 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti - Flag antibody or pulled down with streptavidin - binding beads .

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B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.


D , E . 293T cells were transfected as indicated and immunoprecipitated with an anti - HA antibody . LATS2 immune complexes were subjected to a kinase assay with His - YAP and cold ATP .

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D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.


F . Lats1 - / - ; Lats2 fl / fl ; SV40 mouse embryonic fibroblasts complemented with vector , LATS2 WT or ΔUBA mutant were infected with empty ( control ) or Zeocin - Cre retroviruses and selected with 400 μg / ml of Zeocin for 4 days . Harvested cells were analyzed by Western blotting for the indicated proteins .

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F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.


( B , C ) ( B ) Growth inhibition assay of E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction . ( C ) Growth inhibition assay of E . coli DH10B cells co - expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid . Growth curve was determined at OD600 . Graphs of panels B and C show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison . Different letters indicate statistically different groups of strains ( p value , 4 . 6x10 - 5 and 5 . 19x10 - 8 for panels B and C respectively ) .

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(B, C) (B) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG induction. (C) Growth inhibition assay of E. coli DH10B cells co-expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid. Growth curve was determined at OD600. Graphs of panels B and C show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 4.6x10-5 and 5.19x10-8 for panels B and C respectively).


( A ) Growth inhibition assay of E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG inducible expression . The growth of E . coli was monitored by CFU counting every 1 hr . Data information : Graphs of panels A , show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison .

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(A) Growth inhibition assay of E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants with IPTG inducible expression. The growth of E. coli was monitored by CFU counting every 1 hr. Data information: Graphs of panels A, show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison.


For membrane permeabilization assays , BW25113 WT alone or ∆ lacY ( pYTA - lacZ ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for ( C ) propidium iodide permeability with cells treated with Propidium iodide and Hoechst for detection by fluorescence microscope ( Scale bar = 5 μm ) . For quantification of cells with PI signals , a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI - stained cells / number of Hoechst - stained cells as indicated .

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For membrane permeabilization assays, BW25113 WT alone or ∆lacY(pYTA-lacZ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were carried out for (C) propidium iodide permeability with cells treated with Propidium iodide and Hoechst for detection by fluorescence microscope (Scale bar = 5 μm). For quantification of cells with PI signals, a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI-stained cells / number of Hoechst-stained cells as indicated.


( D ) Bacteriostatic activity assay . E . coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 hr . The IPTG - induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG . Cell density was measured again before continuous growth for additional 1 hr . Data information : Graphs of panels show mean ± SD of three biological replicates ( n = 3 ) , each averaged with 3 technical repeats . One way ANOVA was used for the analysis of statistical significance followed by the Tukey ' s multiple comparison . Different letters indicate statistically different groups of strains ( p value , 1 × 10 - 16 and 2 × 10 - 16 for panels D respectively ) .

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(D) Bacteriostatic activity assay. E. coli DH10B cells harbouring pTrc200 vector or each of its derivatives expressing Tde1 variants were cultured with or without IPTG induction for 1 hr. The IPTG-induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG. Cell density was measured again before continuous growth for additional 1 hr. Data information: Graphs of panels show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 1×10-16 and 2×10-16 for panels D respectively).


( A ) Co - immunoprecipitation ( Co - IP ) in A . tumefaciens . A . tumefaciens C58 ∆ tde1 harbouring pTrc200 vector or its derivatives expressing HA - tagged Tde1 variants . Anti - HA resin was used to coprecipitate the Tde1 variants and Tap1 .

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(A) Co-immunoprecipitation (Co-IP) in A. tumefaciens. A. tumefaciens C58 ∆tde1 harbouring pTrc200 vector or its derivatives expressing HA-tagged Tde1 variants. Anti-HA resin was used to coprecipitate the Tde1 variants and Tap1.


( B ) Secretion assay for HA - tagged Tde1 variants . Western blot for the cellular and extracellular fractions of A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK expressing the HA - tagged Tde1 variants . Hcp secretion was detected as a positive control for active T6SS secretion . Representative western blot results of three biological repeats were shown with antibody against HA , Hcp , or EF - Tu where EF - Tu serve as a loading and non - secreted protein control . Protein markers are indicated in kDa .

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(B) Secretion assay for HA-tagged Tde1 variants. Western blot for the cellular and extracellular fractions of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the HA-tagged Tde1 variants. Hcp secretion was detected as a positive control for active T6SS secretion. Representative western blot results of three biological repeats were shown with antibody against HA, Hcp, or EF-Tu where EF-Tu serve as a loading and non-secreted protein control. Protein markers are indicated in kDa.


( A ) Fluorescence microscopy for Tde1 translocation . A . tumefaciens C58 ∆ tdei expressing Tde1 variants fused with sfGFP ( in green ) and E . coli DH10B carrying mCherry ( false coloured in blue ) were co - cultured for 20 hrs . A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A . tumefaciens to E . coli ( Scale bar = 5 μm ) .

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(A) Fluorescence microscopy for Tde1 translocation. A. tumefaciens C58 ∆tdei expressing Tde1 variants fused with sfGFP (in green) and E. coli DH10B carrying mCherry (false coloured in blue) were co-cultured for 20 hrs. A cyan fluorescence with merged blue and green signals represented the translocation of Tde1 variants from A. tumefaciens to E. coli (Scale bar = 5 μm).


( B ) The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats ( number of cells with cyan fluorescence / total E . coli cells counted ) .

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(B) The number of cells with overlayed GFP and mCherry fluorescence was quantified from a total of 6 randomly selected images obtained from three biological repeats (number of cells with cyan fluorescence/ total E. coli cells counted).


( A ) Interbacterial competition of A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK expressing the Tde1 variants against E . coli cells was carried out on LB medium and E . coli survival rate was quantified by CFU counting . ( B ) Interbacterial competition between various A . tumefaciens C58 strains and A . tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index . Data information : Data in panel A are mean ± SD of four biological repeats of two independent experiments ( n = 4 ) . Panels B show mean ± SD of three biological repeats ( n = 3 ) . One way ANOVA was used for the analysis of statistical significance followed by the Fishers Least Significant Difference ( LSD ) test for panel A and B Different letters indicate statistically different groups of strains ( p value , 3 . 63 × 10 - 4 , 2 . 70 × 10 - 3 , 2 . 3 × 10 - 15 for panels A , B respectively ) .

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(A) Interbacterial competition of A. tumefaciens C58 ∆tdei and ∆tdei∆tssK expressing the Tde1 variants against E. coli cells was carried out on LB medium and E. coli survival rate was quantified by CFU counting. (B) Interbacterial competition between various A. tumefaciens C58 strains and A. tumefaciens 1D1609 on AK medium and the competition outcome was shown by competitive index. Data information: Data in panel A are mean ± SD of four biological repeats of two independent experiments (n=4). Panels B show mean ± SD of three biological repeats (n=3). One way ANOVA was used for the analysis of statistical significance followed by the Fishers Least Significant Difference (LSD) test for panel A and B Different letters indicate statistically different groups of strains (p value, 3.63×10-4, 2.70 × 10-3, 2.3 × 10-15 for panels A, B respectively).


( C ) Secretion assay for Tde1 and its variants co - expressed with its immunity protein Tdi1 in A . tumefaciens C58 ∆ tdei and ∆ tdei ∆ tssK . Data information : Western blots were detected with specific antibody against Tde1 , Hcp , or EF - Tu serving as a loading and non - secreted protein control . Protein markers are indicated in kDa . Results in panel C are representative of three biological repeats .

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(C) Secretion assay for Tde1 and its variants co-expressed with its immunity protein Tdi1 in A. tumefaciens C58 ∆tdei and ∆tdei∆tssK. Data information: Western blots were detected with specific antibody against Tde1, Hcp, or EF-Tu serving as a loading and non-secreted protein control. Protein markers are indicated in kDa. Results in panel C are representative of three biological repeats.


A microfluidic device for quantitative recovery time measurements . Each PDMS - fabricated chip consists of six independent channels , one of which is shown , from top view ( top row ) . Green circles indicate media inflow ; black circle indicates outflow . Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers ( gray circles ) , the height of which ensures that bacteria are imaged in a monolayer ( bottom row ) . Growth conditions ( e . g . antibiotic dose profile ) are controlled via programmable syringe pumps . Fluorescent images ( bottom row ) show representative growth of a monolayer of E . coli BW25113 cells constitutively expressing GFP over four hours .

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A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.


Representative time series fluorescence data showing dose - dependent population recovery in response to transient streptomycin treatment . Here , time zero corresponds to the end of treatment ( 120 minutes ) . Trajectories show mean and standard deviation for five replicates ; colors indicate increasing antibiotic concentration ( 2 , 4 , 6 , 8 , 10 , 12 μg / mL ) . Fluorescence values are normalized to those at time zero . Dotted line indicates a 2 - fold increase , corresponding to the recovery time for each population .

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Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.


Recovery time increases with total antibiotic exposure . Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations ( as in panel C ) . When plotted against total antibiotic exposure ( calculated as ) , all recovery time values collapse onto a single exponential function ( black line , R2 = 0 . 83 ) . Recovery time was measured in response to streptomycin concencentrations of 0 , 2 , 4 , 6 , 8 , 10 , and 12 μg / mL , and treatment durations 30 , 60 , 90 , and 120 minutes .

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Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.


Streptomycin treatment ( closed data points , R2 = 0 . 83 ) results in significantly longer recovery times than chloramphenicol treatment ( open data points ) . Here , the difference between two responses is statistically significant ( p < 0 . 05 by ANOVA ) .

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Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).


The addition of efflux pump inhibitor ( CCCP ) ( closed data points , R2 = 0 . 91 ) increases population recovery time in response to chloramphenicol treatment ( open data points , R2 = 0 . 80 ) . Here , CCCP was added at subinhibitory concentrations ( 3μg / mL ) ; in the absence of antibiotic treatment , CCCP alone did not inhibit population recovery . The CCCP - mediated increase in recovery time is statistically significant ( p < 0 . 01 ) .

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The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).


The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0 . 01 % w / v ( closed data points ) to 0 . 05 % w / v ( open data points ) . Faster synthesis resulted in lower recovery times ( p < 0 . 001 ) in response to streptomycin treatment . In each of these panels , the color scheme indicates increasing antibiotic concentration , as in Figure 1C .

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The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.


Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop . Antibiotics are as follows : penicillin G ( PenG ) , spectinomycin ( Spec ) , gentamicin ( Gent ) , streptomycin ( Str ) , chloramphenicol ( Cm ) , tetracyclin ( Tet ) , and carbenicillin ( Carb ) . Antibiotic concentrations used have been scaled by their respective IC50 values ( solid points and open circles show response with no efflux inhibition and 2μg / mL CCCP , respectively ) . Colors correspond to the motifs of action shown in Figure 4A .

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Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.


A Baseline blood counts of granulocytes . One - way ANOVA ; n = 8 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

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A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


B Flow cytometry quantification of neutrophils in digested hearts . The representative dot plots show the gating strategy for cardiacneutrophils ( CD45 + Ly6G + CD11b + ) at ZT5 . One - way ANOVA ; n = 5 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

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B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


C Representative immunostainings for neutrophils in the myocardium ( left ventricle ) , identified as Ly6G - positive ( 20x magnification ) .

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C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).


D Cardiac mRNA expression levels normalized to HPRT . One - way ANOVA ; n = 5 mice per ZT ; ZT5 vs . ZT17 : * P = 0 . 0064 ( CXCL1 ) , * P = 0 . 0007 ( CXCL2 ) , * P = 0 . 0007 ( CXCL5 ) , * P = 0 . 0001 ( ICAM - 1 ) , * P = 0 . 0009 ( VCAM - 1 ) , * P = 0 . 0181 ( CCL3 ) , * P = 0 . 0360 ( CCL5 ) .

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D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).


E Mean fluorescence intensity ( MFI ) of CXCR2 expression by neutrophils in blood and heart . One - way ANOVA ; n = 3 mice for ZT1 , ZT17 , ZT21 and n = 5 for ZT5 , ZT9 , ZT13 ; ZT5 vs . ZT13 : * P = 0 . 0425 ( blood ) , * P = 0 . 0078 ( heart ) .

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E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).


A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area , identified as Ly6G - positive ( 5x and 20x magnifications ) . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0161 ( 12h MI ) , * P = 0 . 003 ( 24h MI ) .

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A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area, identified as Ly6G-positive (5x and 20x magnifications). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 mice for no MI, n = 5 for 12h post MI, n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0161 (12h MI), *P = 0.003 (24h MI).


B Flow cytometry analysis of neutrophils in bone marrow . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0471 ( 12h MI ) , * P = 0 . 0035 ( 24h MI ) .

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B Flow cytometry analysis of neutrophils in bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice for no MI, n = 5 for 12h post MI, n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0471 (12h MI), *P = 0.0035 (24h MI).


C Blood counts of granulocytes . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 9 mice for no MI , n = 10 for 12h post MI , n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0364 ( 24h MI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Blood counts of granulocytes. Two-way ANOVA followed by Bonferroni post-hoc test; n= 9 mice for no MI, n = 10 for 12h post MI, n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0364 (24h MI).


D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 3 mice for no MI in both ZT groups , n = 5 for ZT5 and n = 6 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0147 ( 24h MI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).


E Representative gating strategy for GMPs in the bone marrow , identified as identified as lineage negative ( CD11b - , Gr1 - , B220 - , CD3 - and Ter119 - ) and Sca - 1 - , c - kit + , CD16 / 32 + , CD34 + . Flow cytometry quantification of GMP in the bone marrow . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 4 mice for no MI and 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative gating strategy for GMPs in the bone marrow, identified as identified as lineage negative (CD11b-, Gr1-, B220-, CD3- and Ter119-) and Sca-1-, c-kit+, CD16/32+, CD34+. Flow cytometry quantification of GMP in the bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for no MI and 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


F CXCL12 levels in bone marrow lavage . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 7 mice for no MI and n = 6 for 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CXCL12 levels in bone marrow lavage. Two-way ANOVA followed by Bonferroni post-hoc test; n = 7 mice for no MI and n = 6 for 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


G Plasma levels of pro - inflammatory cytokines and chemokines . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 11 mice for no MI in both ZT groups , n = 7 for ZT5 and n = 8 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0271 ( CXCL12 , no MI ) , * P = 0 . 0108 ( TNFα , 24h MI ) , * P = 0 . 001 ( G - CSF , 24h MI ) , * P = 0 . 005 ( CXCL1 , 24h MI ) , * P = 0 . 0005 ( CXCL2 , 24h MI ) , * P = 0 . 0016 ( CCL3 24h MI ) , * P = 0 . 0144 ( CCL5 , 24h MI ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plasma levels of pro-inflammatory cytokines and chemokines. Two-way ANOVA followed by Bonferroni post-hoc test; n = 11 mice for no MI in both ZT groups, n = 7 for ZT5 and n = 8 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0271 (CXCL12, no MI), *P = 0.0108 (TNFα, 24h MI), *P = 0.001 (G-CSF, 24h MI), *P = 0.005 (CXCL1, 24h MI), *P = 0.0005 (CXCL2, 24h MI), *P = 0.0016 (CCL3 24h MI), *P = 0.0144 (CCL5, 24h MI).


A Permanent LAD occlusion was perfomed at ZT5 or ZT13 . TTC staining ( white , infarct ; red , vital myocardium ) and quantification of infarct size normalized to the left ventricle ( LV ) . Student ` s t - test ; n = 4 mice for ZT5 and n = 5 for ZT13 MI ; * P = 0 . 0041 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.


B Plasma troponin I levels 24 hours after MI . Student ` s t - test ; n = 4 mice for ZT5 and n = 5 for ZT13 MI ; * P = 0 . 0007 .

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B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007.


C Flow cytometry analysis of dead cardiomyocytes ( CD45 - , zombie + ) 24 hours after MI . Student ` s t - test ; n = 3 mice in both groups ; * P = 0 . 0072 .

O O O O O O O O O O O O O O O O O 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 Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072.


D Survival rates after MI and cause of death . Log - rank test ; n = 87 mice in both groups ; * P = 0 . 0006 .

D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006.


E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin ( αSMA ) staining as ratio between stained and total area of random fields . Student ` s t - test ; n = 4 mice in both groups ; * P = 0 . 0134 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134.


F Masson ´ s trichome staining of fibrosis ( blue , collagen ; red , vital myocardium ) and quantification relative to total LV ( * P = 0 . 0095 ) as well as LV anterior wall thickness ( * P = 0 . 0068 ) 7 days after MI . Student ` s t - test ; n = 6 mice for ZT5 and n = 7 for ZT13 MI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI.


G Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI . Student ` s t - test ; n = 4 mice for ZT5 and n = 5 for ZT13 MI ; * P = 0 . 0175 .

O O O O O O O O O O O O O O O O O O O O O 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 Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175.


H Echocardiography assessment of ejection fraction ( EF ) , end - systolic - volume ( ESV ) and end - diastolic - volume ( EDV ) . Two - way ANOVA ; n = 6 mice for no MI for both groups , n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI , n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI ; ZT5 vs . ZT13 : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0042 ( EF , 7d ) , * P = 0 . 0121 ( EF , 14d ) ; * P = 0 . 0253 ( ESV , 3d ) , * P = 0 . 0421 ( ESV , 7d ) , * P = 0 . 0005 ( ESV , 14d ) ; * P = 0 . 0053 ( EDV , 14d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Echocardiography assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).


B Flow cytometry analysis of cardiacneutrophils 24 hours after ZT13 MI . The dotted line indicates cardiacneutrophil counts 24h after ZT5 MI , shown in Fig . 2A . Student ` s t - test ; n = 3 mice for isotype and n = 5 for Ly6G injected mice ; * P = 0 . 0004 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry analysis of cardiacneutrophils 24 hours after ZT13 MI. The dotted line indicates cardiacneutrophil counts 24h after ZT5 MI, shown in Fig. 2A. Student`s t-test; n = 3 mice for isotype and n = 5 for Ly6G injected mice; *P = 0.0004.


C Plasma TNFα levels 24 hours after ZT13 MI . Student ` s t - test . n = 8 independent samples for isotype and n = 6 for Ly6G injected mice ; * P = 0 . 0019 .

O O O O O O O O O O O O O O O O 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 Plasma TNFα levels 24 hours after ZT13 MI. Student`s t-test. n=8 independent samples for isotype and n=6 for Ly6G injected mice; *P = 0.0019.


D Infarct size relative to left ventricular area ( LV ) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI . Student ` s t - test ; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13 ; Isotype vs . Ly6G : * P = 0 . 0158 ( ZT13 ) . For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI ; * P = 0 . 0012 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Infarct size relative to left ventricular area (LV) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.


E Masson ' s - trichrome staining of fibrosis ( blue , collagen ; red , vital myocardium ) and quantification relative to total area of the LV ( * P = 0 . 0084 ) ; morphometric quantification of the LV anterior wall thickness ( * P = 0 . 0027 ) 7 days after ZT13 MI . Student ` s t - test ; n = 5 mice for isotype and n = 4 mice for Ly6G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Masson's-trichrome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total area of the LV (*P = 0.0084); morphometric quantification of the LV anterior wall thickness (*P = 0.0027) 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G.


F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI . Student ` s t - test ; n = 5 mice for isotype and n = 4 mice for Ly6G ; * P = 0 . 0375 .

F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G; *P = 0.0375.


G Echocardiography measurement of ejection fraction ( EF ) , end - systolic - volume ( ESV ) and end - diastolic - volume ( EDV ) before and after ZT13 MI . Two - way ANOVA ; n = 6 mice for no MI in both groups , n = 9 for isotype and n = 5 for Ly6G at 72h post MI , n = 8 for isotype and n = 4 for Ly6G at 7d post MI . Isotype vs . Ly6G : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0227 ( EF , 7d ) ; MI * P = 0 . 0133 ( ESV , 3d ) , * P = 0 . 0004 ( ESV , 7d ) ; * P = 0 . 0017 ( EDV , 7d ) .

G Echocardiography measurement of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV) before and after ZT13 MI. Two-way ANOVA; n = 6 mice for no MI in both groups, n = 9 for isotype and n = 5 for Ly6G at 72h post MI, n = 8 for isotype and n = 4 for Ly6G at 7d post MI. Isotype vs. Ly6G: *P = 0.0001 (EF, 3d), *P = 0.0227 (EF, 7d); MI *P = 0.0133 (ESV, 3d), *P = 0.0004 (ESV, 7d); *P = 0.0017 (EDV, 7d).


A Representative flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 4 mice for all time points in both groups ; ZT5 vs . ZT13 : * P = 0 . 0001 ( 4h ) , * P = 0 . 0001 ( 8h ) , * P = 0 . 0447 ( 12h ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h).


B Percentage and mean fluorescence intensity ( MFI ) of CXCX2 expression by cardiacneutrophils 12h post - MI after ZT5 and ZT13 MI . Student ` s t - test ; n = 4 mice in both groups ; * P = 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O 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 Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiacneutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001.


C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0005 ( ZT13 ) .

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

C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).


D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0079 ( ZT13 ) .

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

D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).


E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0002 ( ZT13 ) .

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

E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13).


F Mean fluorescence intensity ( MFI ) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype ( WT ) and CXCR2 KO mice . Student ` s t - test . N = 6 mice in both groups ; * P = 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Mean fluorescence intensity (MFI) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype (WT) and CXCR2 KO mice. Student`s t-test. N = 6 mice in both groups; *P = 0.0001.


G Percentage of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice . Two - way ANOVA followed by Bonferroni post - hoc test ; for ZT5 n = 6 mice in both groups , for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice ; WT vs . CXCR2 KO : * P = 0 . 0001 ( ZT13 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0001 (ZT13).


H Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice . Two - way ANOVA followed by Bonferroni post - hoc test ; for ZT5 n = n = 6 mice in both groups , for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice ; WT vs . CXCR2 KO : * P = 0 . 0461 ( ZT13 ) , ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n= n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0461 (ZT13), ns = not significant.


B Percentage of CXCR2hineutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 . 0046 ( ZT13 ) ; ZT5 vs . ZT13 : * P = 0 . 0153 ( DMSO ) .

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 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 O O O O O O O O O O O

B Percentage of CXCR2hineutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0046 (ZT13); ZT5 vs. ZT13: *P = 0.0153 (DMSO).


C Flow cytometry quantification of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 . 0006 ( ZT13 ) ; ZT5 vs . ZT13 : * P = 0 . 0001 ( DMSO ) .

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 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 O O O O O O O O O O O

C Flow cytometry quantification of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0006 (ZT13); ZT5 vs. ZT13: *P = 0.0001 (DMSO).


D Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc test ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 , 0486 ( ZT13 ) .

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

D Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0,0486 (ZT13).


A . Immunoblots of subcellular fractions from HeLa cells exposed to normoxia . P : post - nuclear supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . B . Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia ( 1 % O2 ) for 5 h . P : post - nuclear supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . C . Quantification of the MAM : PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 .

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A. Immunoblots of subcellular fractions from HeLa cells exposed to normoxia. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.B. Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia (1% O2) for 5 h. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.C. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ** P < 0.01.


D . Control HeLa cells or hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Arrows indicate gold particles .

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

D. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Arrows indicate gold particles.


E . Control HeLa cells or hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - TOM20 antibody ( 12 nm gold particles ) . Bar = 500 nm .

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

E. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-TOM20 antibody (12 nm gold particles). Bar = 500 nm.


F . Quantification of the MAM : mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic ( 1 % O2 ) conditions ; Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 60 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) , * * P < 0 . 01 .

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

F. Quantification of the MAM:mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic (1% O2) conditions; Data (mean ± s.e.m.) are from 3 independent experiments. n = 60 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria), ** P < 0.01.


G . Quantification of the MAM : mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic ( 1 % O2 ) conditions . Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 50 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) . NS means not significant .

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

G. Quantification of the MAM:mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic (1% O2) conditions. Data (mean ± s.e.m.) are from 3 independent experiments. n = 50 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria). NS means not significant.


H . The quantification of MAM counts from 50 cells , * * * P < 0 . 001 .

H. The quantification of MAM counts from 50 cells, ***P < 0.001.


I . The quantification of FACL4 immunogold particles from 50 cells , * * * P < 0 . 001 .

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I. The quantification of FACL4 immunogold particles from 50 cells, *** P < 0.001.


J . Hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) and anti - FACL4 ( 6 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Yellow arrows indicate FUNDC1 - gold particles and green arrows indicate FACL4 - gold particles .

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J. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-FACL4 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate FACL4-gold particles.


K . The ratio of FACL4 immunogold particles on the MAM and mitochondrion . n = 80 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) .

O O O O O O O O O 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. The ratio of FACL4 immunogold particles on the MAM and mitochondrion. n = 80 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria).


L . Hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) and anti - DRP1 ( 6 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Yellow arrows indicate FUNDC1 - gold particles and green arrows indicate DRP1 - gold particles .

L. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-DRP1 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate DRP1-gold particles.


B . HeLa cells were transfected with FUNDC1 - MYC or empty vector for 24 h . Cell lysates were immunoprecipitated by anti - MYC antibody and immunoblotted with indicated antibodies .

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B. HeLa cells were transfected with FUNDC1-MYC or empty vector for 24 h. Cell lysates were immunoprecipitated by anti-MYC antibody and immunoblotted with indicated antibodies.


C . HeLa cells were transfected with FLAG - FUNDC1 . About 24 h post - transfection , cell lysates were immunoprecipitated by anti - Calnexin and immunoblotted with anti - FLAG and anti - Calnexin antibodies .

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C. HeLa cells were transfected with FLAG-FUNDC1. About 24 h post-transfection, cell lysates were immunoprecipitated by anti-Calnexin and immunoblotted with anti-FLAG and anti-Calnexin antibodies.


D and E . HeLa cells were transfected with the indicated FUNDC1 - MYC constructs for 24 h . Cell lysates were immunoprecipitated using anti - Calnexin and immunoblotted using anti - MYC and anti - Calnexin antibodies .

O O O O O O O O O O O O O O O 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 and E. HeLa cells were transfected with the indicated FUNDC1-MYC constructs for 24 h. Cell lysates were immunoprecipitated using anti-Calnexin and immunoblotted using anti-MYC and anti-Calnexin antibodies.


F . Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post - nuclear supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated membrane . DRP1 ( S ) and DRP1 ( L ) indicate short and long exposures , respectively . G . Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA ( si - CNX ) and exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post nuclear supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated membrane . DRP1 ( S ) and DRP1 ( L ) indicate short and long exposures , respectively . H . Quantification of the MAM : PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si - CNX and exposed to hypoxia ( 1 % O2 ) for 5 h . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.


A . HeLa cells were transfected with the indicated si - RNAs . 24 h post - transfection , cells were exposed to hypoxia ( 1 % O2 ) for 12 h and then immunostained by anti - TOM20 . Bar = 50 µm . Boxed regions in the left panels are enlarged in the right panels . C . Quantification of mitochondria morphology ( fragmented , intermediate and elongated mitochondria ) in 100 cells from ( A ) . Data represent mean ± s . e . m . ( * * * P < 0 . 001 ) .

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

A. HeLa cells were transfected with the indicated si-RNAs. 24 h post-transfection, cells were exposed to hypoxia (1% O2) for 12 h and then immunostained by anti-TOM20. Bar = 50 µm. Boxed regions in the left panels are enlarged in the right panels.C. Quantification of mitochondria morphology (fragmented, intermediate and elongated mitochondria) in 100 cells from (A). Data represent mean ± s.e.m. (*** P<0.001).


B . Electron microscopy analysis of HeLa cells treated with the indicated si - RNAs for 24 h and then exposed to hypoxia ( 1 % O2 ) for 12 h . Bar = 1 μm . Boxed regions in the left panels are enlarged in the right panels .

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B. Electron microscopy analysis of HeLa cells treated with the indicated si-RNAs for 24 h and then exposed to hypoxia (1% O2) for 12 h. Bar = 1 μm. Boxed regions in the left panels are enlarged in the right panels.


A . HeLa cells were treated with the indicated si - RNAs and exposed to hypoxia for 12 h . Cells were fixed and immunostained by anti - TOM20 and anti - DRP1 antibodies . Bar = 40 µm .

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A. HeLa cells were treated with the indicated si-RNAs and exposed to hypoxia for 12 h. Cells were fixed and immunostained by anti-TOM20 and anti-DRP1 antibodies. Bar = 40 µm.


B . Immunogold electron microscopy of control HeLa cells ( top ) or HeLa cells transfected with si - FUNDC1 ( bottom ) and then exposed to hypoxia ( 1 % O2 ) for 12 h . DRP1 was detected with anti - DRP1 ( 12 nm gold particles ) . Blue stars indicate mitochondria . Yellow arrows indicate gold particles . Red arrows indicate double - membraned autophagosomes .

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B. Immunogold electron microscopy of control HeLa cells (top) or HeLa cells transfected with si-FUNDC1 (bottom) and then exposed to hypoxia (1% O2) for 12 h. DRP1 was detected with anti-DRP1 (12 nm gold particles). Blue stars indicate mitochondria. Yellow arrows indicate gold particles. Red arrows indicate double-membraned autophagosomes.


C - H . Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si - RNA and then exposed to hypoxia for 5 h . PNS : post nuclear supernatant ; Cyto : cytosol ; Mito ( crude ) : crude mitochondrial fraction containing mitochondria and MAMs .

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C-H. Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si-RNA and then exposed to hypoxia for 5 h. PNS: post nuclear supernatant; Cyto: cytosol; Mito (crude): crude mitochondrial fraction containing mitochondria and MAMs.


A . HeLa cells were co - transfected with FLAG - DRP1 and FUNDC1 - MYC for 24 h . Cells were lysed and immunoprecipitated using anti - FLAG antibody . FLAG - DRP1 and FUNDC1 - MYC were analyzed with the corresponding antibodies .

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A. HeLa cells were co-transfected with FLAG-DRP1 and FUNDC1-MYC for 24 h. Cells were lysed and immunoprecipitated using anti-FLAG antibody. FLAG-DRP1 and FUNDC1-MYC were analyzed with the corresponding antibodies.


B . HeLa cells were co - transfected with empty vector , MYC - DRP1 and FLAG - FUNDC1 . 24 h after transfection , cell lysates were immunoprecipitated using anti - FLAG antibody and immunoblotted with anti - MYC and anti - FLAG antibodies .

O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were co-transfected with empty vector, MYC-DRP1 and FLAG-FUNDC1. 24 h after transfection, cell lysates were immunoprecipitated using anti-FLAG antibody and immunoblotted with anti-MYC and anti-FLAG antibodies.


C . MEFs were transfected with FUNDC1 - MYC . 24 h post - transfection , cells were fixed and immunostained by anti - MYC and anti - DRP1 . Bar = 10 µm .

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C. MEFs were transfected with FUNDC1-MYC. 24 h post-transfection, cells were fixed and immunostained by anti-MYC and anti-DRP1. Bar = 10 µm.


D and E . HeLa cells were co - transfected with the indicated FUNDC1 - MYC and FLAG - DRP1 constructs for 24 h . Cell lysates were analyzed by immunoprecipitation using anti - FLAG and immunoblotted with anti - MYC and anti - FLAG antibodies .

O O O O O O O O O O O O O O O O O O O O O O O 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 and E. HeLa cells were co-transfected with the indicated FUNDC1-MYC and FLAG-DRP1 constructs for 24 h. Cell lysates were analyzed by immunoprecipitation using anti-FLAG and immunoblotted with anti-MYC and anti-FLAG antibodies.


F . HeLa cells were exposed to hypoxia ( 1 % O2 ) for the indicated times and cell lysates were immunoprecipitated using anti - FUNDC1 antibody . Endogenous DRP1 , Calnexin , LC3 and FUNDC1 were detected by anti - DRP1 , anti - Calnexin , anti - LC3 , and anti - FUNDC1 antibodies .

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F. HeLa cells were exposed to hypoxia (1% O2) for the indicated times and cell lysates were immunoprecipitated using anti-FUNDC1 antibody. Endogenous DRP1, Calnexin, LC3 and FUNDC1 were detected by anti-DRP1, anti-Calnexin, anti-LC3, and anti-FUNDC1 antibodies.


G . 10 - 20 g GST - FUNDC1 or GST was immobilized on glutathione sepharose resin ( GE Healthcare ) . 20 - 50 g DRP1 was incubated with the GST - FUNDC1 or GST bound resin overnight at 4 ° C . The resin was then extensively rinsed and eluted . Samples were subjected to SDS - PAGE , and then visualized by coomassie blue staining and western blot , respectively .

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G. 10-20 g GST-FUNDC1 or GST was immobilized on glutathione sepharose resin (GE Healthcare). 20-50 g DRP1 was incubated with the GST-FUNDC1 or GST bound resin overnight at 4 °C. The resin was then extensively rinsed and eluted. Samples were subjected to SDS-PAGE, and then visualized by coomassie blue staining and western blot, respectively.


H . Full - length FUNDC1 - MYC , FUNDC1 - MYC ( AA 96 - 155 ) , FL ( Δ129 - 138 ) or vector control were transfected with Mito - DsRed ( red ) into FUNDC1 KD MEFs . 12 h post - transfection , cells were exposed to hypoxia for 12 h and then stained with anti - MYC ( blue ) and anti - DRP1 ( green ) . Bar = 10 µm . Numbers at the right show the average length of mitochondria in Mito - DsRed - positive cells .

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H. Full-length FUNDC1-MYC, FUNDC1-MYC (AA 96-155), FL (Δ129-138) or vector control were transfected with Mito-DsRed (red) into FUNDC1 KD MEFs. 12 h post- transfection, cells were exposed to hypoxia for 12 h and then stained with anti-MYC (blue) and anti-DRP1 (green). Bar = 10 µm. Numbers at the right show the average length of mitochondria in Mito-DsRed-positive cells.


A . HeLa cells were transfected with the indicated si - RNAs for 24 h and then exposed to hypoxia for 24 h or not . Cells were fixed and immunostained by anti - TOM20 and anti - LC3 antibodies . The colocalization of LC3 and mitochondria was quantified and listed on the right side . Bar = 5 µm .

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A. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h or not. Cells were fixed and immunostained by anti-TOM20 and anti-LC3 antibodies. The colocalization of LC3 and mitochondria was quantified and listed on the right side. Bar = 5 µm.


B and C . HeLa cells were transfected with the indicated si - RNAs for 24 h and then exposed to hypoxia for 24 h . Cells were then treated with or without 50 nM BAF A1 ( Bafilomycin A1 ) for 6 h before harvesting . Cell lysates were immunoblotted with the indicated antibodies .

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

B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies.


D and E . Quantification of the TIM23 : Actin and TOM20 : Actin ratios from ( B and C ) . All data are from 3 independent experiments .

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D and E. Quantification of the TIM23:Actin and TOM20:Actin ratios from (B and C). All data are from 3 independent experiments.


F . HeLa cells were transfected by different truncated FUNDC1 - MYC . About 24h post - transfection , cell lysates were immunoblotted using indicated anti - bodies .

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F. HeLa cells were transfected by different truncated FUNDC1-MYC. About 24h post-transfection, cell lysates were immunoblotted using indicated anti-bodies.


A ) Representative specimen of colorectal ( CRC ) primary tumour stained with haematoxylin / eosin / saffron ( HES ) , or antibodies against E - cadherin or Vimentin . i ) The blue , orange and pink dotted lines highlight the normal mucosa , the submucosa and the muscularis propria respectively . Red dotted line highlights the neoplastic tissue . Black arrowheads indicate the direction of invasion . Boxed regions ii and iii show high magnification of normal colonic glands ( ii ) and the CRC invasive front ( iii ) . Scale bar : 2 mm and 500 μm .

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A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.


B ) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin . Boxed regions i , ii and iii are high magnifications of the luminal cavity of normal colonic gland ( i ) and colorectal carcinoma glands ( ii and iii ) . Aarrowheads point to the apical pole enriched in villin . Scale bars : 50 μm .

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B) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin. Boxed regions i, ii and iii are high magnifications of the luminal cavity of normal colonic gland (i) and colorectal carcinoma glands (ii and iii). Aarrowheads point to the apical pole enriched in villin. Scale bars: 50 μm.


( C ) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16

(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients


A ) Representative confocal images of non - invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen ( Ezrin ) , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii , iii and iv are displayed at high magnification . Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars show off - centered nuclei . Scale bars : 20 μm .

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A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.


Graphs representing the percentage of adenocarcinoma explants displaying lumens ( B ) from 8 patients . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per patients . The error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( ns : non significant ) .

Graphs representing the percentage of adenocarcinoma explants displaying lumens (B) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


Graphs representing the percentage of adenocarcinoma explants displaying protrusions ( C ) from 8 patients . Evaluation performed from DIC observation of live tissues , at least 50 explants were analysed per patients . The error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( ns : non significant ) .

Graphs representing the percentage of adenocarcinoma explants displaying protrusions (C) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).


D ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel monitored by DIC microscopy over 2 days Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars point to lumens . Scale bars : 50 μm .

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D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


E ) Images of an invading gland of a colorectal NOS adenocarcinoma , at the light microscopic level ( i , semithin section , 100x objective ) , and as seen by electron microscopy ( ii , 1000x magnification ; and iii , 4000x magnification ) . Note that the bulk of the invading neoplastic gland is in a state of glandular organization ( regions denoted by arrowheads in the left panel ) : epithelia ( E , middle panel ) are polarized to form a lumen ( L ) , and are attached to a basal membrane ( asterisk ) by hemidesmosomes ; the gland is surrounded by fibroblasts ( F ) . However , this organization is focally disrupted at the tubular invasion pole ( arrows in the left panel ) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).


A ) Representative images of Caco - 2 cysts transfected with control siRNA ( siGlo ) or siRNAs targeting ROCK1 and ROCK2 , fixed and stained for prominin - 1 , F - actin ( Phalloidin ) and Nuclei ( Hoechst ) . Confocal Z - sections of cysts are displayed . Arrowheads show non - protruding cells . Arrows point to protruding cells . White stars mark nuclei that are off - centered relative to the cyst ' s monolayer . Scale bars : 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.


B ) Representative confocal images of Caco - 2 cysts treated with ROCK inhibitors Y27632 and H1152 , non - treated ( NT ) or transduced with ROCK1 and ROCK2 shRNAs ( shROCK1 + 2 ) . The cysts were fixed 2 days after invasion and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i and ii are displayed at high magnification . White arrowheads point to non - protruding cells , green arrowheads point to the apical pole , arrows point to protruding cells and white stars show off - centered nuclei . Scale bar : 20 μm .

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

B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.


C ) Bar graph representing the percentage of protrusive Caco - 2 cysts from three independent experiments . Caco - 2 cysts non - treated ( NT ) , incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2 . The cysts were fixed 2 days after invasion , over 100 cysts were counted for each condition . Error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * p < 0 . 001 , * * p < 0 . 01 ) .

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

C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).


E ) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non - treated ( NT ) , in ROCK inhibited conditions ( Y27632 , H1152 ) or in cells transfected with siRNA against ROCK1 and ROCK2 ( siROCK1 + 2 ) . The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , * * p < 0 . 01 ) .

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

E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).


F ) High magnification of boxed region from ( D ) . The top ( i ) and the bottom ( ii ) side of the Boyden chamber membrane were stained for F - actin , E - cadherin , ZO - 1 , Ezrin and Hoechst . Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion . Scale bars : 40 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 O O O O O O O O O O O O O O O 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) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.


A ) Representative images of organoids formed from patient derived xenografts ( PDXs ) that have been embedded in collagen - I gels during 3 days , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) . At end - point , organoids were stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . i and ii are high magnification of boxed regions of + Y27632 and + H1152 organoids respectively . Arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments . Over 50 organoids were counted per condition ( Means ± SEM ) . P values were calculated using paired t - test ( * * * p < 0 . 001 ) .

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

A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001).


C ) Representative images of human adenocarcinoma explants after 2 days in 3D collagen - I gel , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) and stained for the apical pole ( Ezrin ) , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions of NT , + Y27632 , and + H1152 explants are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . D Explants displaying ( D ) lumens from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , n . s : non significant ) .

O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


Explants displaying ( E ) protrusive cells from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , n . s : non significant ) .

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

Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


F ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars point to lumens . Scale bars : 50 μm .

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

F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


A ) Caco - 2 cysts expressing an empty vector ( pLKO ) or shROCK1 ( # 02 ) or shROCK2 ( # 36 ) after 2 days of invasion were fixed and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnifications . Arrowheads point to non - protruding cells . Arrow points to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The number of protrusive Caco - 2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts ( Means ± SEM of at least 3 independent experiments , paired t - test , * * * p < 0 . 001 , * p < 0 . 05 n . s : non significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant).


C ) Caco - 2 cysts stably expressing GFP alone or GFP - ROCK2DN # 2 , were submitted to a 2 days period invasion assay , then fixed and stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . White and black stars show nuclei that engage in protrusions . Scale bars : 20μm . D ) Bargraph representing the percentage of protrusive cysts transduced with GFP ( CTRL , with or without Y27632 ) or with ROCK2DN # 2 ( Means ± SEM of at least 3 independent experiments , paired t - test , * * p < 0 . 01 n . s : non significant ) .

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

C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant).


E ) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole ( arrows ) reminiscent of collective invasion . T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus - ROCK2 - DA and treated with doxycycline 1μg / ml . Scale bars : 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.


F ) Bar graph representing the percentage of protrusive T84 cysts in control ( WT ) , treated with ROCK inhibitor ( Y27632 ) or transduced with lentiviruses expressing GFP or GFP - ROCK2 - DA after induction by Doxycycline ( Means ± SEM of at least 3 independent experiments ) ( paired t - test , * * * p < 0 . 001 , * p < 0 . 05 ) .

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

F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).


G ) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set ( n = 477 patients with survival annotation ) using Kaplan - Meier survival analysis . The patient cohort was divided into tumours with or without a down - regulation of ROCK2 relatively to non - tumoural tissues ( expression cut - off value corresponds to the 1st decile of ROCK2 expression in non - tumoural sample , i . e . 7 . 6 ) . Log - rank test p - values for survival distribution difference are indicated at 3 and 5 years .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.


B ) DIC acquisition of fixed mosaic Caco - 2 cysts expressing GFP alone , GFP - ROCK2DN # 1 or GFP - ROCK2DN # 2 . Boxed regions i and ii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . Scale bars : 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DIC acquisition of fixed mosaic Caco-2 cysts expressing GFP alone, GFP-ROCK2DN#1 or GFP-ROCK2DN#2. Boxed regions i and ii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. Scale bars: 20μm.


C ) Bar graph representing the percentage of mosaic Caco - 2 cysts transduced with GFP or GFP - ROCK2DN # , with or without Y27632 , harbouring a protruding morphology ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * p < 0 . 001 , * p < 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 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

C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).


D ) Quantification of the fate of the GFP positive cells , leader or follower , in mosaic cysts transduced with GFP - alone and treated with Y27632 ( GFP + Y27632 ) or GFP - ROCK2DN # 2 from 3 independent experiments ( Means ± SEM , unpaired t - test , * * * p < 0 . 001 ) .

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

D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).


E ) Representative pull - down of RAC1 - GTP by PAK - Crib , in Caco - 2 cells treated with ROCK inhibitor Y27632 , H1152 or blebbistatin and lysed at 12 or 16 hours . Immunoblot analyses were performed with anti - Rac1 and anti - GAPDH antibody from three independent experiments .

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

E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.


F ) DIC acquisition of fixed Caco - 2 cysts treated with ROCK inhibitors alone ( Y27632 or H1152 ) , or in combination with RAC1 inhibitor NSC23766 . Arrowheads point to non - protruding cells and arrow point to protruding cell . Scale bar 20 μm .

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

F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.


G ) Bar graph representing the percentage of protruding Caco - 2 cysts in control conditions ( non - treated , NT ) or treated with NSC23766 , Y27632 or H1152 ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * * p < 0 . 0001 , * * * 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 B-CONTROLLED_VAR 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

G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05