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 .

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


GPS reporter cells expressing peptide libraries ending with a random ( X ) or Gly ( G ) residue were treated with dominant - negative ( DN ) cullins and analyzed . Data are presented as mean ± standard deviation from nine replicates . GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC - box proteins and analyzed . VHL and FEM1C serve as unrelated BC - box protein controls . Data are presented as mean ± standard deviation from nine replicates .

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

GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.


Comparisons of protein stability among wild - type protein and the mutants indicated on the right .

Comparisons of protein stability among wild-type protein and the mutants indicated on the right.


( A ) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations . The number on each graph denotes the left - tailed p - value of the - 1 residue . CT - inside and CT - outside represent membrane proteins with their C - termini facing the cytosol or extracellular space , respectively .

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(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.


( G ) Confocal images of U2OS cells expressing MTSGLUL - GFP proteins with or without a C - terminal diGly degron . MitoTracker staining shows mitochondria . Scale bar = 20 µm . ( H ) Confocal images of U2OS cells expressing MTSGLUL - GFP - diGly degron fusion proteins co - stained with MitoTracker , and with or without DNCul2 treatment . Scale bar = 20 µm .

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(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.


Stability analysis of indicated proteins in cells treated with shRNAs against various BC - box proteins . Since GFP was tagged at the protein N - terminus and signal peptide and MTSs need to be exposed to be functional , all tested proteins were mislocalized . Protein stability analyses of wild - type ( WT ) and mutant proteins lacking their extreme terminal Gly ( ∆ G ) or having it masked by adding Leu ( + L ) in cells with or without BC - box protein knockdown ( C ) or overexpression ( D ) .

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

Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).


Fractionation and Western blot analysis of wild - type and Flag - tagged PDP2 ( top ) , and wild - type and MTS - deleted ( ∆ MTS ) MRPL28 ( bottom ) . Flag - PDP2 and ∆ MTS MRPL28 were mislocalized . W , C , and M denote whole cell lysates , cytosol fraction and mitochondrial fraction , respectively . Tubulin and VDAC serve as fractionation quality controls .

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Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.


Protein abundance analysis of HA - tagged wild - type or mutant secretory proteins with their signal peptide deleted ( ∆ SP ) in cells with or without KLHDC3 or APPBP2 expression . The HA epitope was inserted immediately after the signal peptide ( SP ) , which had no impact on protein secretion .

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Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.


Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2 , DNCul2 or IMP - 1088 treatments . IMP - 1088 inhibits NMTs . C and M denote cytosol and mitochondrial fractions , respectively . Tubulin and VDAC serve as fractionation quality controls . The relative abundance of cytosolic MIC19 / tubulin was normalized to that of lane 1 and is indicated under each lane in red . Another replicate of this experiment is shown in Fig . EV3B .

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Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.


Abundance comparison of GFP N - or C - terminal tagged with the wild - type or mutant N - terminal ( aa . 1 - 24 ) or C - terminal ( a . a . 218 - 227 ) motif of MIC19 in cells with indicated treatments . Data from sh # 1 is shown here .

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Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.


Fractionation analysis of wild - type HA - MIC19 in U2OS cells treated with IMP - 1088 and with or without inhibition of ZYG11B or KLHDC2 . The normalized relative abundance of cytosolic HA - MIC19 / tubulin is indicated under each lane in red .

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Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.


GST pull - down assay using cells expressing GST or GST - tagged wild - type or mutant KLHDC2 and HA - tagged SDE2 , FAU or TUG .

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GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.


Relative stability analysis of GAPDH C - terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays . The - 3 , - 4 or - 5 residue of SARS - CoV NSP proteins was mutated to Ala ( A ) as indicated . The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2 . Data are presented as the mean value of GFP / RFP + / - standard deviation from triplicate experiments .

Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.


a , Wild - type ( WT ) and Park2 - / - BMDMs were infected with mCherry - expressing M . tuberculosis ( Mtb ) for 4 h and immunostained using anti - parkin antibodies .

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a, Wild-type (WT) and Park2-/- BMDMs were infected with mCherry-expressing M. tuberculosis (Mtb) for 4 h and immunostained using anti-parkin antibodies.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for polyubiquitin .

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b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for polyubiquitin . c , Quantification of ubiquitin - positive M . tuberculosis from b . Results are means ± s . e . m . of three independent experiments ( * * P < 0 . 001 , paired Student ' s t - test ) .

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b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin. c, Quantification of ubiquitin-positive M. tuberculosis from b. Results are means ± s.e.m. of three independent experiments (**P < 0.001, paired Student's t-test).


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h and immunostained for polyubiquitin .

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d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin.


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h and immunostained for polyubiquitin . e , Quantification of ubiquitin positive M . tuberculosis from d , results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , Student ' s t - test ) . f , The parkin and actin expression in cells from e was determined by western blotting .

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d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin. e, Quantification of ubiquitin positive M. tuberculosis from d, results are means ± s.e.m. of three independent experiments (**P  0.005, Student's t-test). f, The parkin and actin expression in cells from e was determined by western blotting.


g , Park2 − / − BMDMs were transduced with lentivirus expressing BFP ( − ) , wild - type parkin , or two separate mutant parkin alleles ( T240R , P437L ) . Cells were infected with M . tuberculosis for 4 h and ubiquitin and M . tuberculosis co - localization was quantified and expressed relative to control BMDMs . Results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , paired Student ' s t - test ) . h , The parkin and actin expression in cells from g was determined by western blotting .

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g, Park2−/− BMDMs were transduced with lentivirus expressing BFP (−), wild-type parkin, or two separate mutant parkin alleles (T240R, P437L). Cells were infected with M. tuberculosis for 4 h and ubiquitin and M. tuberculosis co-localization was quantified and expressed relative to control BMDMs. Results are means ± s.e.m. of three independent experiments (**P  0.005, paired Student's t-test). h, The parkin and actin expression in cells from g was determined by western blotting.


a , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained using linkage - specific ubiquitin antibodies . b , Quantification of K63 and K48 ubiquitin - positive M . tuberculosis from a .

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a, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained using linkage-specific ubiquitin antibodies. b, Quantification of K63 and K48 ubiquitin-positive M. tuberculosis from a.


c , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for NDP52 , p62 , phospho - TBK1 or NBR1 . d , Quantification of co - localization from c ( n = 3 per group , * * P 0 . 001 ) . All errors , s . e . m .

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c, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for NDP52, p62, phospho-TBK1 or NBR1. d, Quantification of co-localization from c (n = 3 per group, **P  0.001). All errors, s.e.m.


a , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 4 h and immunostained for LC3 ( LC3B antibody ) or ATG12 . b , Quantification of results from a ( n = 3 per group , * * P 0 . 001 ) . c , Western blot analysis of LC3 ( LC3B antibody ) from cell lysates from a .

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a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h and immunostained for LAMP1 .

d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h and immunostained for LAMP1 . e , Quantification of results from d expressed relative to control BMDMs ( n = 3 per group , * * P 0 . 001 ) .

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d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001).


f , BMDMs were infected with M . tuberculosis and colony - forming units ( c . f . u . ) at t = 0 and t = 16 h were determined by plating . Results were normalized to t = 0 ( n = 3 per group , * P < 0 . 02 ) .

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f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P < 0.02).


g , RAW264 . 7 macrophages transduced with lentivirus expressing green fluorescent protein ( GFP ) or parkin were infected with M . tuberculosis and c . f . u . were determined after 16 h . Results were normalized to GFP - expressing cells ( n = 3 per group , * * P < 0 . 0076 ) .

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g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P < 0.0076).


h , U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M . tuberculosis for 36 h and c . f . u were determined . Results were normalized to t = 0 ( N = 3 per group , * P < 0 . 02 ) . All errors , s . e . m .

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h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P< 0.02). All errors, s.e.m.


a , WT and Park2 - / - mice were infected with wild - type M . tuberculosis by aerosol and lung bacterial burdens were determined by plating ( means ± s . d . , n = 5 per group , * P < 0 . 02 by Student ' s t - test ) .

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a, WT and Park2-/- mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P < 0.02 by Student's t-test).


b , Enumeration of liver and spleenc . f . u . from mice infected in a , 21 days post - infection ( means ± s . d , n = 5 per group , * P < 0 . 03 by Student ' s t - test ) .

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b, Enumeration of liver and spleenc.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P < 0.03 by Student's t-test).


c , Survival of M . tuberculosis - infected wild - type and Park2 - / - mice ( n = 10 , * * P < 0 . 001 by log - rank test ) .

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c, Survival of M. tuberculosis-infected wild-type and Park2-/- mice (n = 10, **P < 0.001 by log-rank test).


d , Immunohistochemistry staining of lung sections from infected mice21 days post - infection , scale bar , 50 µm .

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d, Immunohistochemistry staining of lung sections from infected mice21 days post-infection, scale bar, 50 µm.


e , Immunohistochemistry staining of lung sections from a human patient with active tuberculosis . Scale bars , 500 µm ( × 4 ) , 50 µm ( × 40 ) .

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e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 µm (×4), 50 µm (×40).


f , Confocal microscopy images of humanlung sections from e immunostained for M . tuberculosis and parkin ; DNA was visualized using DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) . Scale bars , 25 µm .

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f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.


g , Mice were infected with wild - type L . monocytogenes by i . p . injection and bacterial burdens in livers and spleens were determined by plating ( means ± s . d . , n = 7 per group , * P < 0 . 04 by Student ' s t - test ) .

g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P < 0.04 by Student's t-test).


a , Wild - type and two independent parkin - deficient D . melanogaster lines ( parkc00062 , parkf01950 ) were infected with L . monocytogenes by anterior abdomen injection . Atg8 processing was monitored in whole - fly protein lysates by western blotting .

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a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting.


b , L . monocytogenes c . f . u . from a were determined by plating ( means ± s . d . , n = 3 - 5 , * * P < 0 . 001 by Student ' s t - test ) .

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b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test).


c , Survival of D . melanogaster was determined ( n = 33 , * * P < 0 . 001 by log - rank test ) .

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c, Survival of D. melanogaster was determined (n = 33, **P < 0.001 by log-rank test).


d , e , Flies were infected with S . enterica serovar Typhimurium and bacterial c . f . u . ( d ) ( means ± s . d . , n = 3 - 5 per group , * * P 0 . 009 by Student ' s t - test ) and fly survival ( e ) were determined ( n = 59 , * * P 0 . 001 by log - rank test ) .

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d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3-5 per group, **P  0.009 by Student's t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test).


f , Survival of flies infected with M . marinum ( n = 37 , * * P 0 . 0045 by log - rank test ) .

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f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).


Immunofluorescence images of U2OS cells expressing FLAG - HA - FAM134 after 24hrs doxycycline induction stained for HA ( green ) and endogenous calnexin ( CANX ; red ) . Scale bar : 20μm .

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Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) purified GST , wild type GST - LC3s , GST - GABARAPs , GST - Ubiquitin ( Ub ) , GST - Tetra - Ubiquitin , A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type and wild - type or ∆ LIR mutant FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) using GST - LC3B and GST - LC3B F52A - V53A as baits . A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.


Immunofluorescence images of U2OS expressing wild - type or ∆ LIR mutant FLAG - HA - FAM134 proteins after 24hrs doxycycline induction under different conditions ( basal = DMSO , Baf . A1 = 2h 200nM Bafilomycin A1 , EBSS = 2h starvation in EBSS ) Scale bar : 10μm ; staining against FAM134 ( HA ; green ) and endogenous LC3B ( red ) .

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Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).


Small sections of sample pictures of U2OS cells stably expressing the ER - phagy reporter ssRFP - GFP - KDEL . Pictures were obtained by the IncuCyte ® S3 ( 10x ) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time . Scale bar 25µm . Time lapse of representative total image views can be found in Movie EV7 and Movie EV8 .

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Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.


Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction . Autophagy flux was induced and blocked by treatment with Torin1 ( 8hrs ) and Bafilomycin A1 ( Baf . A1 , 8hrs ) , respectively . n =

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Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3


Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts ( MEFs ) .

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Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).


Western blot for Fam134 proteins in indicated MEFs , expressing the ER - phagy reporter ssRFP - GFP - KDEL and being reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein .

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Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.


Profile plots showing the Log2 LFQ intensity ( G ) and representative immunofluorescence ( H ) of Climp63 ( green ) in wild - type and Fam134 knockout MEFs .

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Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs.


Profile plots showing the Log2 LFQ intensity ( I ) and representative immunofluorescence ( J ) for collagen I ( red ) expression in wild - type and Fam134 knockout MEFs . Scale bar : 10μm . Inset scale bar : 5μm . Nuclei were stained with Hoechst 33342 .

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Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.


Electron microscopy of wild - type and Fam134 single knockout MEFs ( ER = endoplasmic reticulum ) . Scale bar : 500nm .

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Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.


Immunofluorescence images stained for endogenous Calnexin ( Canx ) or Climp63 in wild - type ( WT ) , Fam134 knockout MEFs , and Fam134 knockout MEFs reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein . Scale bar : 10μm . Inset scale bar : 5μm .

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Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.


Representative Western blots of total cell lysate derived from MEFs used in ( B ) and detected for endogenous Fam134 and Actin , respectively .

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Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively.


Representative Western Blot of Collagen I in wild - type MEFs and Fam134 knockout MEFs reconstituted for the respective wild - type ( WT ) or ∆ LIR mutant Fam134 protein . Vinculin has been used as reference for ratio calculation . The statistical significance was estimated by unpaired Student ' s t test . All data are represented as mean ± s . e . m . of n = 3 biological experiments and the statistical significance is defined as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.


Representative Western Blot ( D ) and the relative bar plot ( E ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆ LIR Fam134a protein .

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Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.


Representative Western Blot ( F ) and the relative bar plot ( G ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c . Representative Western Blot ( H ) and the relative bar plot ( I ) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b .

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Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.


Representative Western Blot ( J ) and the relative bar plot ( K ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b . Representative Western Blot ( L ) and the relative bar plot ( M ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c .

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Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.


C . Confocal images of MDA - MB - 231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D - collagen matrix . Cells were imaged and stained with Phalloidin - 568 ( red ) , pMLCII / Alexa ‑ 633 ( false ‑ coloured green ) or imaged for ZsGreen ( false - coloured white ) . Scale 20 μm .

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C. Confocal images of MDA-MB-231 cells transfected with ZsGreen or ZsRASSF1C grown in 3D-collagen matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for ZsGreen (false-coloured white). Scale 20 μm.


D . Single cell morphology assay of MDA - MB - 231 cells cultured in 3D - collagen and analysed for morphology 24 hours after transient knock - down using either a control sequence ( NT , non - targeting ) or a sequence targeting the RASSF1 locus . RASSF1 knock - down marks a reduction in rounded cells . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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D. Single cell morphology assay of MDA-MB-231 cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


C . Western blot analysis of proteins in MDA - MB - 231 cells transiently transfected with siRNA targeting a control sequence ( NT ) or targeting RASSF1 . GAPDH was used as a loading control .

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C. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control.


D . Immunoprecipitation from MDA - MB - 231 transiently transfected with control ( pcDNA3 ) or HA ‑ RASSF1C plasmid . Pull ‑ down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies .

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D. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA‑RASSF1C plasmid. Pull‑down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies.


H . Quantification of rounded versus elongated cells in single cell morphology assay in 3D ‑ collagen indicating the degree of mesenchymal - amoeboid transition of MDA - MB - 231 cells when RASSF1C or the described mutants are expressed . I . Representative confocal images show elongated or rounded cells per field of view of MDA ‑ MB - 231 cells transfected with DsRed , DsRASSF1C , DsRASSF1C - R197W or DsRASSF1C - R199F , grown in 3D ‑ collagen and stained with Phalloidin - 568 ( red ) for F ‑ actin and pMLCII / Alexa 633 . Scale bars represent 10 μm .

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H. Quantification of rounded versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. I. Representative confocal images show elongated or rounded cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and stained with Phalloidin-568 (red) for F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm.


A . Left : Cartoon showing the principle behind the Cre - carrying EVs and how recipient cells switch from DsRed + to eGFP + expression upon Cre - mediated excision of the DsRed locus . Centre : representative images , and right : quantification of T47DDsRed cells that converted to eGFP + in the bottom wells of a transwell system in the presence of MDA - MB - 231CFP ; Cre ; Control or MDA ‑ MB ‑ 231CFP ; Cre ; HA - RASSF1C seeded in the upper wells . Scale bars represent 100 μm . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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A. Left: Cartoon showing the principle behind the Cre-carrying EVs and how recipient cells switch from DsRed+ to eGFP+ expression upon Cre-mediated excision of the DsRed locus. Centre: representative images, and right: quantification of T47DDsRed cells that converted to eGFP+ in the bottom wells of a transwell system in the presence of MDA-MB-231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C seeded in the upper wells. Scale bars represent 100 μm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.


C . Western blot analysis of EVs isolated from MDA - MB - 231 cells transfected with Control ( pcDNA3 ) or FLAG - RASSF1C with indicated antibodies . Equal amounts of protein were loaded after normalization via microBCA assay .

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C. Western blot analysis of EVs isolated from MDA-MB-231 cells transfected with Control (pcDNA3) or FLAG-RASSF1C with indicated antibodies. Equal amounts of protein were loaded after normalization via microBCA assay.


F . Left : Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells , treated with 20 ng / μl EVs derived from MDA - MB - 231 cells , transfected the same constructs used in ( e ) . Right : Quantification of Boyden chamber invasion 24 hours after EV treatment . Scale bars represent 200µm . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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F. Left: Representative images of Boyden chamber invasion assay in 3D matrigel of MCF7 cells, treated with 20 ng/μl EVs derived from MDA-MB-231 cells, transfected the same constructs used in (e). Right: Quantification of Boyden chamber invasion 24 hours after EV treatment. Scale bars represent 200µm. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.


D . Representative confocal images ( upper row ) and quantification of fluorescent intensity of ALDH1 expression in MDA - MB - 231 cells expressing RASSF1C ( black bar in the graph ) or after siRNA treatment against RASSF1 ( grey bar ) . Lower row shows merged images with DAPI ( blue ) , ALDH1 ( green ) and RASSF1C ( red ) . Scale bars represent 20 μm . Data Information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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D. Representative confocal images (upper row) and quantification of fluorescent intensity of ALDH1 expression in MDA-MB-231 cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1 (grey bar). Lower row shows merged images with DAPI (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm. Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


J . qRT ‑ PCR expression of NANOG , OCT4 , SOX2 in MCF7 cells 24 h after exposure to 50 ng / μl EVs derived from MDA ‑ MB ‑ 231 cells ( treated as indicated ) . Data Information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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J. qRT‑PCR expression of NANOG, OCT4, SOX2 in MCF7 cells 24 h after exposure to 50 ng/μl EVs derived from MDA‑MB‑231 cells (treated as indicated). Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.


B . Left : representative confocal images of mammary tumor formed by the co - injection of T47DDsRed reporter cells with either MDA - MB - 231CFP ; Cre ; Control or MDA - MB - 231CFP ; Cre ; HA - RASSF1C . Scale 200 μm . Right : averaged values for all mice for the percent of the eGFP + or CFP + cells as part of the whole tumor . Data information : All data are from n = 3 independent experiments . Data are analyzed by Student T - test and represented as mean ± SEM . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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B. Left: representative confocal images of mammary tumor formed by the co-injection of T47DDsRed reporter cells with either MDA-MB-231CFP;Cre;Control or MDA-MB-231CFP;Cre;HA-RASSF1C. Scale 200 μm. Right: averaged values for all mice for the percent of the eGFP+ or CFP+ cells as part of the whole tumor. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.


B . Kaplan - Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter / CpG island methylation of RASSF1A , previously reported to be associated with outcome in breast cancer patients . P values were obtained from a log - rank test .

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B. Kaplan-Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter/CpG island methylation of RASSF1A, previously reported to be associated with outcome in breast cancer patients. P values were obtained from a log-rank test.


C . Left : heatmap depicting methylation patterns across the RASSF1 gene . In light green , RASSF1 - 2γ CpG island is depicted ; in dark green , RASSF1 - 1α CpG island is depicted . Right : Kaplan ‑ Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients . P values were calculated from a log - rank test . Color legend : yellow , low RASSF1A and low RASSF1C ; green , low RASSF1A and high RASSF1C ; red , high RASSF1A and low RASSF1C ; grey , high RASSF1A and high RASSF1C .

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C. Left: heatmap depicting methylation patterns across the RASSF1 gene. In light green, RASSF1-2γ CpG island is depicted; in dark green, RASSF1-1α CpG island is depicted. Right: Kaplan‑Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients. P values were calculated from a log-rank test. Color legend: yellow, low RASSF1A and low RASSF1C; green, low RASSF1A and high RASSF1C; red, high RASSF1A and low RASSF1C; grey, high RASSF1A and high RASSF1C.


Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2 - ( I401Q ) mutant compared to that of wild - type Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

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Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2- (I401Q) mutant compared to that of wild-type Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 - 3 LNS2SS2 - and LNS2SS2 - ( I401Q ) , plotted on a logarithmic scale . Error bars represent SEM and replicate numbers are indicated in bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 .

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Comparison of binding affinities for Nxph1 binding to mouse Nrxn1-3 LNS2SS2- and LNS2SS2- (I401Q), plotted on a logarithmic scale. Error bars represent SEM and replicate numbers are indicated in bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01.


Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A + and Nrxn1 LNS2SS2AB + compared to that of Nrxn1 LNS2SS2 - . Replicate numbers are shown in E .

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Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E.


Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice - insert containing Nrxn1 LNS2 with the I401Q mutation ; error bars represent SEM and replicate numbers are indicated in or above bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.


Representative fluorescence polarization binding curves are shown for LNS2SS2 - and LNS2SS2A + interacting with Nxph1 with or without Ca2 + added .

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Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added.


Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2 - and Nrxn1 LNS2SS2A + with or without Ca2 + added ; replicate numbers are indicated in bars . Error bars represent SEM , and significance values were calculated using Welch ' s t - test . ns , not significant .

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Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.


Comparison of binding affinities of Nxph1 binding to different Nrxn1 - 3 LNS2 isoforms .

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Comparison of binding affinities of Nxph1 binding to different Nrxn1-3 LNS2 isoforms.


Comparison of binding affinities for Nxph3 binding to Nrxn1 - 3 LNS2SS2 - .

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Comparison of binding affinities for Nxph3 binding to Nrxn1-3 LNS2SS2-.


Sucrose gradient UV profile of 60S / 40S / IAPV - IRES reaction mixture after an overnight run . The peaks corresponding to 80S and 40S were used for RNA extraction and UREA - PAGE shown in the inset .

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Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset.


Representative cryo - EM image where roughly half of the particles correspond to 40S ( blue ) and the other half to 80S ( orange ) .

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Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).


( H , I ) Induction of Ctcflos and Ucp1 in vivo during cold - induced iWAT browning . Relative expression levels of ( H ) Ctcflos transcript 1 and ( I ) Ucp1 in iWAT of C57BL / 6J mice held at 5 ° C for one week compared to age and weight matched mice held at 30 ° C or 23 ° C , assessed by quantitative PCR ( qPCR ) . Mean and individual values , n = 7 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.


( D ) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 1 ( biological replicate ) for Ucp1n = 3 ( biological replicates ) for Ctcflos .

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(D) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice, assessed by qPCR. Mean values ± SD, n=1 (biological replicate) for Ucp1n=3 (biological replicates) for Ctcflos .


( E ) Ctcflos , 45SrRNA and 12SrRNA transcript abundance in nuclear , cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 6 ( biological replicates ) , two - way ANOVA ( Tukey - test ) , * * * * p < 0 . 0001 .

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(E) Ctcflos, 45SrRNA and 12SrRNA transcript abundance in nuclear, cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice, assessed by qPCR. Mean values ± SD, n=6 (biological replicates), two-way ANOVA (Tukey-test), ****p< 0.0001.


( E ) Relative Ucp1 transcript levels in response to Ctcflos tr1 , 3 and 4 KD by ASO 1 , 3 , 4 and 2 , 5 6 , respectively , compared to nontargeting controls , assessed by qPCR . Mean values ± SD , n = 3 - 4 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(E) Relative Ucp1 transcript levels in response to Ctcflos tr1, 3 and 4 KD by ASO 1, 3, 4 and 2, 5 6, respectively, compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), ***p<0.001, ****p<0.0001.


( F ) Relative expression levels of cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) and cytochrome c oxidase subunit 7a1 ( Cox7a1 ) in Ctcflos tr1 , 3 and 4 KD compared to control samples , assessed by qPCR . Mean values ± SD , n = 3 ( biological replicates ) - 4 , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) Relative expression levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) and cytochrome c oxidase subunit 7a1 (Cox7a1) in Ctcflos tr1, 3 and 4 KD compared to control samples, assessed by qPCR. Mean values ± SD, n=3 (biological replicates)-4, unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( H ) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 ( UCP1 ( red ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) ) .

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(H) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining by DAPI (green); phase contrast (PC) (gray)).


Impact of Ctcflos KD on mitochondrial biogenesis . ( R ) Microscopic images of Mito Tracker stained brite adipocytes ( Mito tracker ( blue ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) )

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Impact of Ctcflos KD on mitochondrial biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green); phase contrast (PC) (gray))


( D ) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 ( Ucp1 ) , cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) , cytochrome c oxidase subunit 7a1 ( Cox7a1 ) , peroxisome proliferator activated receptor γ coactivator 1α ( Pgc1a ) and elongation of very long chain fatty acids - like 3 ( Elovl3 ) , comparing Ctcflos tr1 ( ASO 1 ) and tr 3 and 4 ( ASO 2 ) KD with their respective controls . Mean values ± SD , n = 6 ( biological replicates ) , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) n . s . p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(D) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome c oxidase subunit 7a1 (Cox7a1), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a) and elongation of very long chain fatty acids-like 3 (Elovl3), comparing Ctcflos tr1 (ASO 1) and tr 3 and 4 (ASO 2) KD with their respective controls. Mean values ± SD, n=6 (biological replicates), unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test) n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( K - M ) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression . Primary iWAT cells infected with Prdm16 - or turbo green fluorescent protein ( GFP ) - expressing viral particles at the second day of proliferation , followed by reverse transfection at the first day of differentiation using nontargeting control ( NC ) or Ctcflos - targeting ASO1 . Gene expression analyzed 72 hours later by qPCR ( K ) Transcript levels of Ctcflos tr1 relative to Fabp4 ( L ) Transcript levels of Prdm16 relative to Fabp4 ( M ) Transcript levels of Ucp1 relative to Fabp4 . Mean and individual values . Each graph presents pooled data from two experiments slightly varying virus titers . RM One - way ANOVA ( Tukey - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(K-M) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression. Primary iWAT cells infected with Prdm16- or turbo green fluorescent protein (GFP)-expressing viral particles at the second day of proliferation, followed by reverse transfection at the first day of differentiation using nontargeting control (NC) or Ctcflos-targeting ASO1. Gene expression analyzed 72 hours later by qPCR (K) Transcript levels of Ctcflos tr1 relative to Fabp4 (L) Transcript levels of Prdm16 relative to Fabp4 (M) Transcript levels of Ucp1 relative to Fabp4. Mean and individual values. Each graph presents pooled data from two experiments slightly varying virus titers. RM One-way ANOVA (Tukey-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( B - D ) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis , ( B ) expressed as Ucp1 relative to Fabp4 mRNA levels , ( C ) Ucp1 relative to Gtf2b and ( D ) Fabp4 relative to Gtf2b . Mean values ± SD , n = 3 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(B-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Effect of Ctcflos KD on Serine / Arginine rich Splicing factor 2 ( SC35 / SRSF2 ) and nuclear speckle abundance in Ctcflos tr1 ( ASO 1 ) KD and control cells ( KD day 1 of differentiation , analysis after 48h ) . ( E ) Immunocytochemistry of SC35 . SC35 signal ( blue ) alone ( middle images ) and SC35 ( blue ) , DNA staining by DAPI ( green ) and phase contrast ( PC ) ( gray ) combined ( outer images ) .

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Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images).


Influence of Ctcflos KD on Prdm16 alternative splicing . ( O ) Relative Prdm16 total , ( P ) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD , comparing Ctcflos tr1 ( ASO1 ) and tr3 , 4 ( ASO2 ) KD and control samples , assessed by qPCR , Mean values ± SD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Influence of Ctcflos KD on Prdm16 alternative splicing. (O) Relative Prdm16 total, (P) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001.


Influence of Ctcflos KD on Prdm16 alternative splicing . ( R , S ) Fold change of Prdm16 short and Prdm16 long ( R ) ASO1 to NC and ( S ) ASO2 and NC 24 hours after Ctcflos KD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * * p < 0 . 01 .

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Influence of Ctcflos KD on Prdm16 alternative splicing. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 and NC 24 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, ** p<0.01.


B Chromatin accessibility as measured by the number of all ( TSS and distal ) ATAC - Seq peaks ( read count ≥ 10 ) detected in DN2 ( n = 5 ) , DN3 ( n = 5 ) , ISP ( n = 5 ) , DPCD3 - ( n = 6 ) , DPCD3 + ( n = 6 ) , SPCD4 + ( n = 5 ) , SPCD8 + ( n = 5 ) populations . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 0 . 035 ( Kruskal - Wallis Test ) .

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B Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test).


C Fraction of OCRs having increasing , decreasing , steady and fluctuating accessibility pattern during T - cell development ( x - axis ) . Average number of normalized reads in OCRs ( y - axis ) .

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C Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7 stages

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A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements database

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B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements

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A The rationale of footprinting analysis as illustrated by actual ATAC - Seq peaks , HINT - ATAC footprint hits , and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6 .

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A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.


B Heatmap of ln ( odds ratio ) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions . TFs with an SD of the motif counts < 10 are excluded .

B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.


D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T - cell precursors and in T - ALLs , respectively ( n = 10 / 19 patients have two biological replicates , n = 9 / 19 patients have one replicate ) . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 4 . 4e - 08 ( Kruskal - Wallis Test ) .

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D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T-cell precursors and in T-ALLs, respectively (n=10/19 patients have two biological replicates, n=9/19 patients have one replicate). Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=4.4e-08 (Kruskal-Wallis Test).


E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T - ALLs in comparison to healthy T - cell precursors . Patients on the x - axis ; same order as in panel ( C ) . Gray cells represent TFs having non - significant enrichment or depletion .

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E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T-ALLs in comparison to healthy T-cell precursors. Patients on the x-axis; same order as in panel (C). Gray cells represent TFs having non-significant enrichment or depletion.


C log2 fold changes of 292 genes which are differentially expressed ( x - axis , RNA - Seq : non - sorted bulk thymi vs . T - ALLs ) and differentially accessible ( y - axis , ATAC - Seq : sorted T - cell populations vs . T - ALLs ) in T - ALLs in comparison to healthy T - cell precursors . Dot size and color indicate the frequency ( number of patients having dysregulation of the gene ) . Genes with ≥ 5 / 19 frequency are shown on the plot . Protein coding genes with ≥ 12 / 19 frequency are labelled . For plotting the average LFC values of ATAC and RNA are used per gene per quartile . Q1 - 4 stands for the quadrant 1 - 4 .

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C log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4.


D Heatmap of VST normalized read counts ( each row is also normalized by the row mean ) of differentially expressed genes ( pval < 0 . 1 ; DESeq2 ) in three patients in comparison to remaining 16 . Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts .

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D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.


( B ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs without heparin .

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(B) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.


( C ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs and heparin .

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(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.


( D ) Bar graph representing residual activated factor X ( FXa ) activity ( ΔOD / min ) in presence of the sdAbs and heparin .

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(D) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.


( B - C ) Graphs reporting the recovery of thrombin - and FXa - mediated substrate conversion in the presence of increasing doses of KB - AT - 23 , in presence of antithrombin and heparin .

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(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.


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( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 .

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(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.


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( B ) Purified KB - AT - 23 - fus and KB - hFX - 11 - fus were given intravenously to wild - type C57B6 mice ( 10 mg / kg ) . At indicated time - points , blood was collected and residual protein was measured . Plotted is residual protein versus time after injection . Green circles : KB - AT - 23 - fus ; Red circles : KB - hFX - 11 - fus .

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(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.


( D ) Graph reporting the volume of blood loss ( µL ) in treated mice ( n = 4 - 9 per group ) observed during 30 minutes post TVT . Dosing was 10 mg / kg for KB - AT - 23 and 1 mg / kg for FVIIa . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) .

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(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).


( B ) Western blot analysis of KB - AT - 23 protein secretion in conditioned media 72 hours post - transfection of HuH7 cells . In the lower panel the western blot quantification via densitometry analysis is depicted . Data are presented as mean ± SD , and were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . * * * * p < 0 . 001 .

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(B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.


( C ) HuH7 - celles were collected 72 hours post - transfection . Presented is the Western blot analysis of lysed cells for the presence of KB - AT - 23 . Cntr - refers to non - transfected cells , while Cntr + refers to purified recombinant KB - AT - 23 .

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(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.


( B ) Representative western blot analyses of KB - AT - 23 and antithrombin expression on plasma samples ( n = 4 ) collected from animals 27 weeks post - AAV injection . kDa , molecular weight marker .

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(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.