Figure 2A 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 ) . 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 ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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).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).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).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).


Figure 3A 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A 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.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.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.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).


Figure 4B 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 . 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 . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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.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.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).


Figure 5A 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 . 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A 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.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).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.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.


Figure 6B 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B 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).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).


Figure 1A . 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 . 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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.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.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.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.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.


Figure 2B . 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 . 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 B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.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.


Figure 3A . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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.


Figure 4A . 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 . 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 . 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 . 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.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.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.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.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.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.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.


Figure 5A . 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 . 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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.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.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.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.


Figure 6A . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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.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.


Figure 1A ) 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 . ( C ) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 1A) 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.(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients


Figure 2A ) 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 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) 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 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.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).


Figure 3A ) 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 . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) 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.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).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.


Figure 4A ) 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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).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.


Figure 5A ) 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 ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) 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). 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).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.


Figure 6B ) 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 ) . 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 . 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 .

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Figure 6B) 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).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.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<0.05


Figure 7A ) siRNA screen performed in Caco - 2 cysts treated with Y27632 : Caco - 2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days . Then , Caco - 2 cysts were treated with Y27632 and the serum gradient was induced . After 2 days , the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining . The bar graph represents the mean invasion rate . Each condition was duplicated . The red dashed line represent the median invasion rate , the red full line indicates the 3 fold decrease in invasion rate . Three hits were identified : # 2 : ARHGEF18 + RNEF , # 7 : SGEF + ARHGEF16 and # 24 : FARP1 + FARP2 ( Mean ± SD ) . B ) Caco - 2 cysts stably expressing empty vector ( pGIPZ ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632 , allowed to invade for 2 days , fixed and stained for F - actin ( Phalloidin ) . Boxed regions i , ii , iii and iv 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 . C ) Bar graphs representing the percentage of protrusive cysts formed from Caco - 2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments ( Means ± SEM , paired t - test , ns : non significant , * * p < 0 . 01 ) . D ) Representative acquisition of Caco - 2 cells overexpressing GFP - FARP2 grown in 2D and stained with ZO - 1 , in non - treated ( NT ) and after 2h with Y27632 or H1152 . The dashed lines crossing the cell - cell junctions ( i , ii and iii ) are representative of the linescans measurements . Scale bars : 20μm . E ) Representative fluorescence intensity of ZO - 1 ( red ) staining and GFP - FARP2 ( green ) along the dashed line i , ii and iii represented corresponding to non - treated , Y27632 or H1152 treatments ) F ) Bar graph representing the average ratio of GFP - FARP2 over ZO - 1 fluorescence intensities measured with linescans crossing cell - cell junctions . The quantification was performed on 71 cells expressing GFP - FARP2 - FL from 3 independent experiments . For each cell , between 10 and 15 lines ( 12 pixels wide ) were drawn across random region of cell - cell junctions positive for ZO - 1 staining ( Mean ± SEM Unpaired t - test * * p < 0 . 01 , * p < 0 . 05 ) .

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Figure 7A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv 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. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01). D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).


Figure 8A ) Caco - 2 cysts stably expressing GFP ( treated or not with Y27632 ) , or Cherry - FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH 730 / 733 ( FARP2 - del / Mut ) were allowed to invade for 2 days , fixed and stained for F - actin . Boxed regions i , ii , iii , iv and v are shown at high magnification . Arrowheads point to non - protruding cells , arrow points to protruding cells , white star shows nucleus that engages in protrusion . Scale bars : 20μm . B ) Bargraph representing the percentage of protrusive cysts expressing GFP ( treated or not with Y27632 ) or the mentioned GFP - FARP2 constructs from 3 independent experiments ( Means ± SEM , paired t - test , * * p < 0 , 01 , n . s : non significant ) . C ) Representative images of Caco - 2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin - IIA and allowed to invade for 2 days . After fixation , the cysts were stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Representative Y27632 - induced « invasive » phenotype and Blebbistatin - induced « dendritic » phenotype are displayed . Arrows point to protruding cells , white star shows nucleus that engages in protrusion and arrowheads point to " dendritic " protrusions . D ) Bar graph representing the percentage of cysts with the " invasive " or " dendritic " phenotypes after treatment with Y27632 , Blebbistatin or siMyosin - IIA or siMyosin - IIB from at least 3 independent experiments ( Means ± SEM , unpaired t - test , * p < 0 , 05 , n . s : non significant ) . E ) Mature Caco - 2 cysts were transduced with GFP or Cherry tagged FARP2 - full length ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH / 730 / 733 ( FARP2 - del / Mut ) , and treated ( or not , NT ) with Blebbistatin . 2 days after invasion , the cysts were fixed and stained for F - actin . Boxed regions i , ii , iii and iv are shown at high magnification . Arrowheads point to non - protruding cells ; arrow points to protruding cells and white stars show nucleus that engage in protrusions . Scale bars : 20 μm . F ) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin , from at least 3 independent experiments ( Means ± SEM , t - test , * * p < 0 , 01 , n . s : non significant ) .

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Figure 8A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant). C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant). E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).


Figure 1G IP of UbcH7 by PknG or PknGΔUbl in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknGΔUbl Mtb strains as in B . H IP of UbcH7 by PknG or PknG E190A in U937 cells . Cells were infected with WT , ΔpknG , or ΔpknG : pknG E190A Mtb strains as in B .

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Figure 1G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B. H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.


Figure 2E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His - UBA1 at 37 ° C for 30 min . The reaction products were analyzed by immunoblotting with antibodies against Ub ( top panel ) , the His - tag on UBA1 ( middle panel ) , and PknG ( bottom panel ) , or by staining with Coomassie Brilliant Blue ( bottom ) . For reducing conditions , samples were treated with 500 mM β - mercaptoethanol ( βME ) prior to SDS - PAGE gel analysis .

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Figure 2E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis.


Figure 3A MS / MS analysis of UbcH7 in the absence ( top ) or presence ( bottom ) of PknG to reveal ubiquitination sites . Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high - confidence peptides possessing di - Gly modifications . The ubiquitinated residue ( Lys82 ) in the peptide sequence is highlighted in red . The peak heights are the relative abundances of the corresponding fragmentation ions . Matched amino terminus - containing ions ( b ions ) are indicated in red , and matched carboxyl terminus - containing ions ( y ions ) are indicated in blue . Only the major peaks that were identified are labeled .

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Figure 3A MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.


Figure 4A Luciferase assay of NF - κB activation in HEK293T cells transfected with WT or mutant Mtb PknG . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way analysis of variance ( ANOVA ) using GraphPad Prism 7 . D Survival of Mtb strains in primary human monocyte - derived macrophages treated as in B and C . The graph shows the mean ± SEM of three independent biological replicates , n = 3 . * P < 0 . 05 , * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 .

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Figure 4A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.


Figure 5B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells . Cells were infected with the indicated Mtb strains at an MOI of 1 . Non - infected cells were used as controls . After 4 hours , the cells were lysed and immunoprecipitated with the antibody against PknG . The immunoprecipitated proteins were immunoblotted with TRAF2 , TAK1 , TAB2 ( negative control ) , or TAB3 ( negative control ) antibodies . E Immunoblotting of TRAF2 , TAK1 , and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours . Cells were treated with CHX ( 50 μg / mL ) with or without MG132 ( 5 μM ) . Cell lysates were analyzed by immunoblotting for TRAF2 ( anti - TRAF2 ) , TAK1 ( anti - TAK1 ) , and PknG ( anti - PknG ) . GAPDH was used as the loading control . Densitometric quantification of immunoblots is indicated below the immunoblots .

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Figure 5B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.


Figure 6G , H In vivo ubiquitination assay of TRAF2 ( G ) and TAK1 ( H ) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours . Cells were lysed and immunoprecipitated using the antibody against Flag . Immunoprecipitated proteins were immunoblotted with the antibody against HA .

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Figure 6G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.


Figure 7A , B qPCR analysis of Tnf mRNA ( A ) and Il6 mRNA ( B ) in splenic cells from C57BL / 6 mice infected with WT or mutant Mtb stains for 0 - 20 days . Data are representative of one experiment with two independent biological replicates ( mean ± SEM of n = 6 mice per group ) . * P < 0 . 05 and * * P < 0 . 01 . Data were analyzed according to one - way ANOVA using GraphPad Prism 7 . D H & E staining of lungs and livers of C57BL / 6 mice treated as in A . Arrows indicate puncta of cellular infiltration . Scale bars , 200 μm .

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Figure 7A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.


Figure 1B , HeLa cells were treated with DMSO or 2µM Mito - C for 15 min and immunostained with anti - TOM20 ( green ) antibody and DAPI ; cropped areas show the mitochondria morphology changes . C , Quantification of mitochondria morphology ( based on TOM20 immunostaining as shown in B ) from HeLa cells treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as ratio of fragmented mitochondria ( percentage of total ) . Errors bars show the standard error of the mean ( SEM ) ( n = 100 , technical replicates ) . E , Time lapse video - microscopy snapshot on HeLa cells transfected with NAF - 1 - mRFP ( red ) and treated with fluoMito - C ( green ) . The distance / intensity fluorescence quantification graph illustrates the codistribution of fluoMito - C and NAF - 1 - mRFP .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.