Figure 1C - D : Confocal images ( C ) and quantification ( D ) of immunofluorescence staining of STAT5b , actin filaments ( phalloidin ) and nuclei ( DAPI ) in cardiomyocytes of cryosections from the hearts of mice transduced with either control - or ERBB4ECD - AAV . Panel D depicts quantification of colocalization of STAT5b - and DAPI - specific signals . One dot in the boxplot corresponds to the median value in 20 - 50 images of stained sections from one heart ( control AAV : n = 4 , ERBB4ECD - AAV : n = 5 , biological replicates ) . Two - tailed unpaired T - test was used for statistics . Scale bar 10µm . White arrows point to cardiomyocyte nuclei . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . E : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in the heart of mice transduced with either control - or ERBB4ECD - AAV . One dot corresponds to analysis of one heart ( n = 10 ; biological replicates combined from two replicate experiments ) . Two - tailed unpaired T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . F - G : Confocal images ( F ) and quantification ( G ) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes . The cells were treated with either control or Stat5b - targeting shRNAs and stimulated with NRG - 1 for two days . Myosin immunoreactivity was used to quantify cell area . One dot in the boxplot corresponds to one cell ( Control no NRG - 1 : n = 83 , control with NRG1 : n = 73 , Stat5b # 1 with NRG - 1 : n = 80 , Stat5b # 2 with NRG1 : n = 75 ; technical replicates combined from four biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA was used . Post hoc analyses were conducted with the Mann Whitney U - test and the resulting P - values corrected with the method of Benjamini , Krieger and Yekutieli . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . H - I : Confocal images ( H ) and quantification ( I ) of immunofluorescence staining of STAT5b and nuclei ( DAPI ) in mouse cardiomyocytes . The cells were treated with either control or Erbb4 - targeting shRNAs . Panel I depicts quantification of colocalization of STAT5b - and DAPI - specific signals ( left ) and the cell perimeter ( right ) . One dot in the boxplots corresponds to one cell ( Control : n = 24 , Erbb4 # 1 : n = 26 , Erbb4 # 2 : n = 35 ; technical replicates combined from two biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA and Dunn ' s multicomparison test ( colocalization analysis ) and Brown - Forsythe one - way ANOVA and Dunnett ' s multicomparison test ( cardiomyocyte size analysis ) with multiple test correction were used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . J : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in mouse cardiomyocytes . The cells were treated with either control , Stat5b - or Erbb4 - targeting shRNAs and stimulated with NRG - 1 for 30 minutes . One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation ( for each group n = 3 ; biological replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test with multiple test correction was used for statistics . * , P ≤ 0 . 05 ; * * P < 0 . 01 ; * * * , P < 0 . 001 against control shRNA ( Igf - 1 panel : Stat5b # 1 P = 0 . 0038 , Stat5b # 2 P = 0 . 0153 , Erbb4 # 1 P = 0 . 0188 , Erbb4 # 2 P = 0 . 0288 ; Myc panel : Stat5b # 1 P = 0 . 0093 , Stat5b # 2 P = 0 . 0119 , Erbb4 # 1 P = 0 . 0197 , Erbb4 # 2 P = 0 . 0512 ; Cdkn1a panel : Stat5b # 1 P = 0 . 0091 , Stat5b # 2 P = 0 . 0107 , Erbb4 # 1 P = 0 . 0235 , Erbb4 # 2 P = 0 . 048 ) . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1C-D: Confocal images (C) and quantification (D) of immunofluorescence staining of STAT5b, actin filaments (phalloidin) and nuclei (DAPI) in cardiomyocytes of cryosections from the hearts of mice transduced with either control- or ERBB4ECD-AAV. Panel D depicts quantification of colocalization of STAT5b- and DAPI-specific signals. One dot in the boxplot corresponds to the median value in 20-50 images of stained sections from one heart (control AAV: n = 4, ERBB4ECD-AAV: n=5, biological replicates). Two-tailed unpaired T-test was used for statistics. Scale bar 10µm. White arrows point to cardiomyocyte nuclei. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.E: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in the heart of mice transduced with either control- or ERBB4ECD-AAV. One dot corresponds to analysis of one heart (n = 10; biological replicates combined from two replicate experiments). Two-tailed unpaired T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.F-G: Confocal images (F) and quantification (G) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either control or Stat5b-targeting shRNAs and stimulated with NRG-1 for two days. Myosin immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (Control no NRG-1: n = 83, control with NRG1: n=73, Stat5b#1 with NRG-1: n=80, Stat5b#2 with NRG1: n=75; technical replicates combined from four biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA was used. Post hoc analyses were conducted with the Mann Whitney U-test and the resulting P-values corrected with the method of Benjamini, Krieger and Yekutieli. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.H-I: Confocal images (H) and quantification (I) of immunofluorescence staining of STAT5b and nuclei (DAPI) in mouse cardiomyocytes. The cells were treated with either control or Erbb4-targeting shRNAs. Panel I depicts quantification of colocalization of STAT5b- and DAPI-specific signals (left) and the cell perimeter (right). One dot in the boxplots corresponds to one cell (Control: n = 24, Erbb4#1: n=26, Erbb4#2: n=35; technical replicates combined from two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and Dunn's multicomparison test (colocalization analysis) and Brown-Forsythe one-way ANOVA and Dunnett's multicomparison test (cardiomyocyte size analysis) with multiple test correction were used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.J: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in mouse cardiomyocytes. The cells were treated with either control, Stat5b- or Erbb4-targeting shRNAs and stimulated with NRG-1 for 30 minutes. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group n = 3; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P ≤ 0.05; ** P < 0.01; ***, P < 0.001 against control shRNA (Igf-1 panel: Stat5b#1 P = 0.0038, Stat5b#2 P = 0.0153, Erbb4#1 P = 0.0188, Erbb4#2 P = 0.0288; Myc panel: Stat5b#1 P = 0.0093, Stat5b#2 P = 0.0119, Erbb4#1 P = 0.0197, Erbb4#2 P = 0.0512; Cdkn1a panel: Stat5b#1 P = 0.0091, Stat5b#2 P = 0.0107, Erbb4#1 P = 0.0235, Erbb4#2 P = 0.048). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Figure 2A - B : Proximity ligation assay ( PLA ) of association of dynamin - 2 with ERBB4 in primary mouse cardiomyocytes treated with either DMSO or the dynamin inhibitor dynasore for 5 hours . Panel A depicts a z projection of a stack of confocal images . PLA interactions are shown in red , nuclear stain DAPI in blue . Panel B depicts quantification of the data . One dot in the boxplot corresponds to the number of PLA signals in µm2 in one cell ( Control : n = 6 , DMSO buffer : n = 11 , dynasore : n = 11 ; technical replicates from one of two biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA and the Dunn ' s multicomparison test with multiple test correction was used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . G : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in primary mouse cardiomyocytes treated for 1 - 3 hours with the DMSO buffer , the ERBB kinase inhibitor AG1478 , or dynasore , and stimulated or not for 30 minutes with NRG - 1 . One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation ( for each group in Igf - 1 and Cdkn1a panel : n = 6 , for each group expect the dynasore group in Myc panel : n = 4 , for the dynasore group in Myc panel : n = 3 ; biological replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test with multiple test correction was used for statistics . * , P < 0 . 05 ; * * P < 0 . 01 ; * * * , P < 0 . 001 ; against DMSO + NRG - 1 treatment ( Igf - 1 panel : DMSO without NRG - 1 P = 0 . 0041 , AG1478 P = 0 . 0082 , dynasore P = 6 . 61e - 06 ; Myc panel : DMSO without NRG - 1 P = 0 . 0386 , AG1478 P = 0 . 0037 , dynasore P = 0 . 0064 ; Cdkn1a panel : DMSO without NRG - 1 P = 0 . 0123 , AG1478 P = 0 . 0064 , dynasore P = 0 . 0092 ) . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . H - I : Confocal images ( H ) and quantification ( I ) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes . The cells were treated with either DMSO or dynasore and stimulated with NRG - 1 for two days . The myosin immunoreactivity was used to quantify cell area . One dot in the boxplot corresponds to one cell ( DMSO : n = 22 , dynasore : n = 28 ; technical replicates combined from three biological replicate experiments ) . Non - parametric Mann - Whitney U - test was used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

Figure 2A-B: Proximity ligation assay (PLA) of association of dynamin-2 with ERBB4 in primary mouse cardiomyocytes treated with either DMSO or the dynamin inhibitor dynasore for 5 hours. Panel A depicts a z projection of a stack of confocal images. PLA interactions are shown in red, nuclear stain DAPI in blue. Panel B depicts quantification of the data. One dot in the boxplot corresponds to the number of PLA signals in µm2 in one cell (Control: n = 6, DMSO buffer: n=11, dynasore: n=11 ; technical replicates from one of two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and the Dunn's multicomparison test with multiple test correction was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.G: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in primary mouse cardiomyocytes treated for 1-3 hours with the DMSO buffer, the ERBB kinase inhibitor AG1478, or dynasore, and stimulated or not for 30 minutes with NRG-1. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group in Igf-1 and Cdkn1a panel: n = 6, for each group expect the dynasore group in Myc panel: n=4, for the dynasore group in Myc panel: n=3 ; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P < 0.05; ** P < 0.01; ***, P < 0.001; against DMSO + NRG-1 treatment (Igf-1 panel: DMSO without NRG-1 P = 0.0041, AG1478 P = 0.0082, dynasore P = 6.61e-06; Myc panel: DMSO without NRG-1 P = 0.0386, AG1478 P = 0.0037, dynasore P = 0.0064; Cdkn1a panel: DMSO without NRG-1 P = 0.0123, AG1478 P = 0.0064, dynasore P = 0.0092). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.H-I: Confocal images (H) and quantification (I) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either DMSO or dynasore and stimulated with NRG-1 for two days. The myosin immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (DMSO: n = 22, dynasore: n = 28; technical replicates combined from three biological replicate experiments). Non-parametric Mann-Whitney U-test was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Figure 3Confocal images ( A and quantification ( B , of immunofluorescence staining of myosin heavy chain ( green ) and DAPI ( blue ) in the hearts of 4 dpf zebrafish embryos injected with either the buffer control or NRG - 1 into the pericardial sac at 2 dpf . The myosin immunoreactivity was used to quantify both the average thickness of the ventricular wall as well as the cross - sectional area of the ventricles . The number of nuclei was estimated with the DAPI stain . The amount nuclei ( red ) in the ventricle and the ventricle area ( yellow ) was quantified to differentiate between hypertrophic and hyperplastic growth . One dot in the boxplots corresponds to one heart ( Control ( A - B ) : n = 10 , NRG - 1 ( A - B ) : n = 7 biological replicates from two replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Scale bars 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . Western analysis of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . ( Control : n = 15 , NRG - 1 : n = 16 ; biological replicates combined from three replicate experiments ) . densitometric quantification of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 3 - 4 zebrafish embryos ( Control : n = 15 , NRG - 1 : n = 16 ; biological replicates combined from three replicate experiments ) . Unpaired two - tailed T - test with Welch ' s correction ( pStat5 ) or unpaired two - tailed T - test ( pErk , pAkt ) was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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 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

Figure 3Confocal images (A and quantification (B, of immunofluorescence staining of myosin heavy chain (green) and DAPI (blue) in the hearts of 4 dpf zebrafish embryos injected with either the buffer control or NRG-1 into the pericardial sac at 2 dpf. The myosin immunoreactivity was used to quantify both the average thickness of the ventricular wall as well as the cross-sectional area of the ventricles. The number of nuclei was estimated with the DAPI stain. The amount nuclei (red) in the ventricle and the ventricle area (yellow) was quantified to differentiate between hypertrophic and hyperplastic growth. One dot in the boxplots corresponds to one heart (Control (A-B): n = 10, NRG-1 (A-B): n = 7 biological replicates from two replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bars 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.Western analysis of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments).densitometric quantification of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 3-4 zebrafish embryos (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments). Unpaired two-tailed T-test with Welch's correction (pStat5) or unpaired two-tailed T-test (pErk, pAkt) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Figure 4Western analysis ( C ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . ( DMSO ( Stat5 ) n = 13 , AG1478 ( Stat5 ) n = 7 , Lapatinib ( Stat5 ) : n = 4 , Gefitinib ( Stat5 ) n = 6 , DMSO ( Akt ) n = 9 , AG1478 ( Akt ) n = 3 , Lapatinib ( Akt ) n = 3 , Gefitinib ( Akt ) n = 3 , DMSO ( Erk ) n = 13 , AG1478 ( Erk ) n = 6 , Lapatinib ( Erk ) n = 4 , Gefitinib ( Erk ) n = 8 ; biological replicates combined from three replicate experiments ) . densitometric quantification ( D ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos ( DMSO ( Stat5 ) n = 13 , AG1478 ( Stat5 ) n = 7 , Lapatinib ( Stat5 ) : n = 4 , Gefitinib ( Stat5 ) n = 6 , DMSO ( Akt ) n = 9 , AG1478 ( Akt ) n = 3 , Lapatinib ( Akt ) n = 3 , Gefitinib ( Akt ) n = 3 , DMSO ( Erk ) n = 13 , AG1478 ( Erk ) n = 6 , Lapatinib ( Erk ) n = 4 , Gefitinib ( Erk ) n = 8 ; biological replicates combined from three replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test ( pStat5 , pAkt ) or non - parametric Kruskal - Wallis with Dunn ' s multicomparison test ( pErk ) was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . E - F : Confocal images ( E ) and quantification ( F ) of immunofluorescence staining of myosin heavy chain in hearts of 4 dpf zebrafish embryos treated with DMSO or dynasore for 2 days . The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross - sectional area of the ventricles . One dot in the boxplots corresponds to one heart ( DMSO ( thickness , ejection fraction ) n = 11 , dynasore ( thickness , ejection fraction ) n = 12 , DMSO ( area ) n = 15 , dynasore ( area ) n = 14 ; biological replicates ) . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . I - J : Western analysis ( I ) and densitometric quantification ( J ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in E . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos ( for each group n = 5 ; biological replicates ) . Unpaired two - tailed T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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 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 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 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

Figure 4Western analysis (C) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments).densitometric quantification (D) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments). One-way ANOVA and the Dunnett's multicomparison test (pStat5, pAkt) or non-parametric Kruskal-Wallis with Dunn's multicomparison test (pErk) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.E-F: Confocal images (E) and quantification (F) of immunofluorescence staining of myosin heavy chain in hearts of 4 dpf zebrafish embryos treated with DMSO or dynasore for 2 days. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. One dot in the boxplots corresponds to one heart (DMSO (thickness, ejection fraction) n = 11, dynasore (thickness, ejection fraction) n = 12, DMSO (area) n = 15, dynasore (area) n = 14; biological replicates). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.I-J: Western analysis (I) and densitometric quantification (J) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in E. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (for each group n = 5; biological replicates). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Figure 5A - B : Confocal images ( A ) and quantification ( B ) of immunofluorescence staining of myosin heavy chain and Stat5b in hearts of 4 dpf zebrafish embryos injected with CRISPR / Cas9 and either control or stat5b - targeting gRNA at one - cell stage . The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross - sectional area of the ventricles . Stat5b staining intensity was also quantified . One dot in the boxplots corresponds to one heart ( control gRNA : n = 14 , stat5b gRNA n = 13 ; biological replicates from one of three replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values . quantification of the ejection fraction ( D ) . The 4 dpf embryos were treated as in A . Ejection fraction was quantified from the videos . One dot in the boxplots corresponds to ejection fraction of one heart ( for each group n = 22 ; biological replicates from one of three replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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 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

Figure 5A-B: Confocal images (A) and quantification (B) of immunofluorescence staining of myosin heavy chain and Stat5b in hearts of 4 dpf zebrafish embryos injected with CRISPR/Cas9 and either control or stat5b-targeting gRNA at one-cell stage. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. Stat5b staining intensity was also quantified. One dot in the boxplots corresponds to one heart (control gRNA: n = 14, stat5b gRNA n = 13; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.quantification of the ejection fraction (D). The 4 dpf embryos were treated as in A. Ejection fraction was quantified from the videos. One dot in the boxplots corresponds to ejection fraction of one heart (for each group n = 22; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Figure 6C - D : Immunohistochemical analysis ( C ) and quantification ( D ) of STAT5 activation in sections representing normal myocardia or pathological cardiac hypertrophy ( n = 3 , aortic stenosis ; n = 2 , idiopathic cardiomyopathy ) . Phospho - STAT5b staining intensity was quantified . One dot in the boxplot corresponds to one subject ( normal : n = 6 , hypertrophic n = 13 ; biological replicates ) . In the boxplot the central band represents the median , the box the interquartile range and whiskers the whole range of values . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm .

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

Figure 6C-D: Immunohistochemical analysis (C) and quantification (D) of STAT5 activation in sections representing normal myocardia or pathological cardiac hypertrophy (n = 3, aortic stenosis; n = 2, idiopathic cardiomyopathy). Phospho-STAT5b staining intensity was quantified. One dot in the boxplot corresponds to one subject (normal: n = 6, hypertrophic n = 13; biological replicates). In the boxplot the central band represents the median, the box the interquartile range and whiskers the whole range of values. Unpaired two-tailed T-test was used for statistics. Scale bar 50µm.


Figure 1 ( A ) Live imaging of MTOC dynamics during meiosis I . Z - projection images ( top ) show MTOCs ( mNG - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Central MTOCs are magnified ( squares ) . 3D - reconstructed spindles are aligned with the spindle axis ( middle ) . Central MTOCs are indicated with arrowheads . Time in h : mm . The kymograph ( bottom ) shows projected signals on the spindle axis for all time points . ( B ) Density maps of MTOCs ( left ) and chromosomes ( right ) along the spindle axis in a representative oocyte . The color represents the percentage of MTOCs or chromosome volumes , respectively , coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) . ( C ) Average density maps of 21 oocytes mapped as in ( B ) . ( D ) Dynamics of MTOC sorting and chromosome congression . Ratio of MTOCs ( left ) or chromosomes ( right ) in the polar region versus those in the middle region of the spindle are plotted over time . ( E ) Dynamics of spindle elongation . The spindle was visualized with EGFP - Map4 The length / width ratio of the spindle was measured after 3D reconstruction ( n = 13 oocytes from 3 independent experiments ) . ( F ) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle . Oocytes were fixed and stained for MTOCs ( pericentrin , green ) , chromosomes ( Hoechst33342 , magenta ) and microtubules ( α - tubulin , grey ) . n = 21 , 28 , 24 oocytes . A representative oocyte fixed at 5 hours after NEBD is shown . Arrowheads indicate central MTOCs .

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 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 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

Figure 1(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points.(B) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). (C) Average density maps of 21 oocytes mapped as in (B). (D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time.(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.


Figure 2 ( A ) Live imaging of MTOC and kinetochore dynamics . MTOCs ( mNG - Cep192 , green ) and kinetochores ( tdTomato - CENP - C , magenta ) in B6D2F1 oocytes are shown . Time in h : mm after NEBD . Circles indicate central MTOCs , which are positioned in the middle region of the spindle later than 4 hours after NEBD . Arrows indicate displacement of the MTOCs . Scale bar , 10 μm . ( B ) Tracking analysis of central MTOCs . In the left graph , the positions of central MTOCs along the spindle axis are shown over time . MTOC positions were used to calculate distance to the spindle equator . In the right graph , lines showing temporal changes in MTOC - equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle . ( C ) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle . trajectories of central MTOCs ( n = 25 MTOCs of 8 oocytes from 3 independent experiments ) were analyzed and their temporal changes in MTOC - equator distance are shown ( Left graph ) . Central MTOCs were categorized into two groups : ( 1 ) ones that came from polar regions of the spindle ( n = 21 , middle graph ) and ( 2 ) others that stayed in the middle region throughout the period before reaching a position closest to the equator ( n = 4 , right graph ) . ( E ) Destination of central MTOCs . The tracks of central MTOCs that originated from polar regions were used ( n = 21 MTOCs ) . The pole of origin was defined as Pole 1 , while the other pole was defined as Pole 2 . MTOC positions along the spindle axis ( positive values for those closer to Pole 1 ) over time are shown . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle . Note that central MTOCs moved back to the original pole ( Pole 1 ) or switched their positions to the opposite pole ( Pole 2 ) . ( F ) Destination of central MTOCs that originated from a polar region was categorized as in ( E ) .

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 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 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 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

Figure 2(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm.(B) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.(C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph).(E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). (F) Destination of central MTOCs that originated from a polar region was categorized as in (E).


Figure 3 ( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . Z - projection and 3D - reconstructed images show MTOCs ( mNeonGreen - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Time in h : mm after NEBD . Scale bar , 10 μm . ( B ) Ndc80 - 9D is defective for MTOC sorting . The density map of MTOCs along the spindle axis in Zp3 - Cre Ndc80f / f oocytes expressing Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . The color represents the percentage of MTOC volume coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) . ( C ) MTOCs do not accumulate at the spindle poles in Ndc80 - 9D - expressing oocytes . Ratio of MTOCs ( green ) or chromosomes ( magenta ) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD ( n = 19 , 19 and 22 oocytes , respectively , from at least 3 independent experiments ) . ( D ) Ndc80 - 9A decreases central MTOCs . The number of central MTOCs ( MTOCs that were positioned in the middle region of the spindle , was determined at each time point ( n = 19 , 21 oocytes , respectively , from at least 3 independent experiments ) .

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 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

Figure 3(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm.(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).(C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments).(D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle , was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments).


Figure 4 ( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , - 9D or - 9A . Z - projection and 3D - reconstructed images show microtubules ( EGFP - Map4 , green ) and chromosomes ( H2B - mCherry , magenta ) . Scale bar , 10 μm . ( B ) Ndc80 - 9D is defective for limiting spindle elongation . The aspect ratio ( length / width ) of 3D - reconstructed spindles was measured over time ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) . ( C ) Ndc80 - 9D causes excessive spindle elongation . Spindle lengths measured after 3D reconstruction are shown ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) .

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 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

Figure 4(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm.(B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).(C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


Figure 5 ( A ) K - fibers are connected to MTOCs and extend to spindle poles . Oocytes 6 hours after NEBD were fixed following cold treatment . Microtubules ( α - tubulin , green ) , MTOCs ( pericentrin , magenta ) , kinetochores ( ACA , red ) and DNA ( Hoechst333342 ) are shown . Five oocytes from 2 independent experiments were analyzed . Scale bar , 10 μm . ( B ) A majority of MTOCs are attached to K - fibers . The volumes of individual MTOCs were measured after 3D reconstruction . The sum volume of MTOCs attached to K - fibers relative to the total volume of MTOCs was calculated for each oocyte ( n = 5 oocytes from 2 independent experiments ) . ( C ) K - fibers extend to spindle poles . The length of K - fibers and distance between kinetochores and a spindle pole ( KT - to - Pole distance ) were measured . K - fiber length relative to the KT - to - Pole distance was calculated ( n = 90 K - fibers of 5 oocytes from 2 independent experiments ) .

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 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

Figure 5(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.(B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments).(C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments).


Figure 2B Measurement of 2 - NBDG uptake in different cell lines by flow cytometry ( n = 4 biological replicates ) . C Quantitative RT - PCR detection of glycolytic gene expression levels in HaCaT cells upon miR - 31 overexpression . β - actin was used as a house - keeping control gene ( n = 3 biological replicates ) G and H Glycolysis stress test of HaCaT cells upon UK5099 ( G , n = 4 biological replicates ) or PC siRNA ( si - PC , H , n = 5 biological replicates ) in 1mM glucose condition . ECAR and OCR were normalized to the amount of protein .

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Figure 2B Measurement of 2-NBDG uptake in different cell lines by flow cytometry (n=4 biological replicates).C Quantitative RT-PCR detection of glycolytic gene expression levels in HaCaT cells upon miR-31 overexpression. β-actin was used as a house-keeping control gene (n=3 biological replicates)G and H Glycolysis stress test of HaCaT cells upon UK5099 (G, n=4 biological replicates) or PC siRNA (si-PC, H, n=5 biological replicates) in 1mM glucose condition. ECAR and OCR were normalized to the amount of protein.


Figure 3B Western blot analysis of GLS , GS , GOT1 , GOT2 , AGC1 in HaCaT cells upon miR - 31 overexpression , Tubulin α was used as loading control . C Violin plot of glutamine consumption , glutamate secretion , and cystine consumption of HaCaT cells upon different treatments ( n = 12 biological replicates ) . G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course ( n = 3 biological replicates ) .

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Figure 3B Western blot analysis of GLS, GS, GOT1, GOT2, AGC1 in HaCaT cells upon miR-31 overexpression, Tubulin α was used as loading control.C Violin plot of glutamine consumption, glutamate secretion, and cystine consumption of HaCaT cells upon different treatments (n=12 biological replicates).G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course (n=3 biological replicates).


Figure 4B Cell viability of HaCaT cell was measured by MTS assay ( n = 4 biological replicates ) . C Quantitative RT - PCR detection of Has - miR - 31 - 5p expression levels in HaCaT cells exposed to a range of glucose ( n = 4 biological replicates ) . RNU6 - 1 was used as house control gene . D Western blot analysis of GS in HaCaT cells with miR - 31 overexpression under different concentrations of glucose or 2 - DG treatment , and Tubulin α was used as a loading control . G and H FACS analysis of Th17 differentiation upon TFB - TBOA treatment ( G , n = 4 biological replicates ) or AOA and iGOT1 treatments ( H ) , n ≥ 3 biological replicates in each group .

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Figure 4B Cell viability of HaCaT cell was measured by MTS assay (n=4 biological replicates).C Quantitative RT-PCR detection of Has-miR-31-5p expression levels in HaCaT cells exposed to a range of glucose (n=4 biological replicates). RNU6-1 was used as house control gene.D Western blot analysis of GS in HaCaT cells with miR-31 overexpression under different concentrations of glucose or 2-DG treatment, and Tubulin α was used as a loading control.G and H FACS analysis of Th17 differentiation upon TFB-TBOA treatment (G, n=4 biological replicates) or AOA and iGOT1 treatments (H), n≥3 biological replicates in each group.


Figure 5A Representative picture of in situ hybridization ( ISH ) staining of miR - 31 in skin biopsies of healthy individuals and psoriasis patients ( n = 5 individuals in each group ) . Quantification of results showing the average level of miR - 31 in skin epidermis ( right panel ) . B , basal layer ; S + G , spinous and granular layers . Scale bars : 100 μm . B Representative pictures of Immunohistochemical staining of GLUT1 , GLS , and GS in skin biopsies of HC and Ps ( n = 5 individuals in each group ) . Scale bars : 200 μm . Disease activity was evaluated by epidermal thickness ( E ) n = 5 mice in Vehicle and n = 6 mice in other groups . H FACS analysis of Th17 cells proportion in splenic lymphocytes of mice ( n = 5 mice in Vehicle and n = 6 mice in other groups ) . I Enzyme - linked immunosorbent assay for the determination of IL - 17A levels in serum of mice ( n = 5 mice in Vehicle and n = 6 mice in other groups ) . J Quantitative RT - PCR detection of mmu - miR - 31 - 5p expression levels in skin of mice ( n = 5 mice in each group ) .

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Figure 5A Representative picture of in situ hybridization (ISH) staining of miR-31 in skin biopsies of healthy individuals and psoriasis patients (n=5 individuals in each group). Quantification of results showing the average level of miR-31 in skin epidermis (right panel). B, basal layer; S+G, spinous and granular layers. Scale bars: 100 μm.B Representative pictures of Immunohistochemical staining of GLUT1, GLS, and GS in skin biopsies of HC and Ps (n=5 individuals in each group). Scale bars: 200 μm.Disease activity was evaluated by epidermal thickness (E) n=5 mice in Vehicle and n=6 mice in other groups.H FACS analysis of Th17 cells proportion in splenic lymphocytes of mice (n=5 mice in Vehicle and n=6 mice in other groups).I Enzyme-linked immunosorbent assay for the determination of IL-17A levels in serum of mice (n=5 mice in Vehicle and n=6 mice in other groups).J Quantitative RT-PCR detection of mmu-miR-31-5p expression levels in skin of mice (n=5 mice in each group).


Figure 1 ( A ) Immunoblotting for Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of AR - 97Q , AR - 24Q , and wild - type mice . ( B ) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up - regulated in AR - 97Q mice ( n = 3 ) . Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) . ( C ) Quantification of the Dnmts mRNA levels of the spinal cord in wild - type , AR - 24Q , and AR - 97Q mice using RT - qPCR ( n = 3 ) . ( D and E ) Representative immunohistochemical images depicting the expression of Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of male AR - 97Q , AR - 24Q , and wild - type mice and quantification of the immunoreactivity for Dnmts ( n = 25 neurons from three mice of each group ) . ( F ) Double - immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR - 97Q mouse . ( G ) Immunoblotting for Dnmt1 of the skeletal muscle of wild - type and AR - 97Q mice . ( H ) Quantification of the signal intensity of Dnmt1 bands ( n = 3 ) .

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Figure 1(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).


Figure 2 ( A ) Immunoblotting of NSC34 cells expressing AR - 24Q or AR - 97Q ( NSC24Q and NSC97Q , respectively ) that were treated with DHT for Dnmt1 , Dnmt3a and Dnmt3b , and quantification of the signal intensities of bands corresponding to Dnmt1 , Dnmt3a and Dnmt3b ( n = 3 ) . ( B ) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT - qPCR ( n = 3 ) . ( C ) Protein levels of Dnmt1 , Dnmt3a and Dnmt3b in DHT - treated and - untreated NSC97Q cells by Western blotting analysis . ( D ) RT - qPCR analysis of Dnmt1 in DHT - treated and - untreated NSC97Q cells ( n = 3 ) . ( E , F ) Quantitative assessment of cell viability changes due to the siRNA - mediated knock down of Dnmt1 , Dnmt3a or Dnmt3b in NSC97Q cells with DHT ( E ) or without DHT ( F ) using the WST - 8 assay ( n = 3 ) . ( G ) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST - 8 assay ( n = 3 per group ) . ( H ) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1 , Dnmt3a and Dnmt3b . ( I , J ) Cell viability analysis with siRNA - mediated knockdown of Dnmts in DHT - untreated NSC97Q ( I ) and DHT - treated NSC24Q ( J ) .

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Figure 2(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).


Figure 3 ( A - D ) Grip power ( A ) , body weight ( B ) , rotarod performance ( C ) ( Two - way ANOVA with Tukey ' s test ) and survival rate ( D ) ( Kaplan - Meier analysis and log - rank test ) of AR - 97Q mice treated with or without RG108 ( DMSO , n = 20 ; 0 . 5 mg / dL , n = 20 ; 2 . 0 mg / dL , n = 20 ) . All parameters were significantly improved in AR - 97Q mice treated with 2 . 0 mg / dL RG108 compared with those treated with DMSO : * P < 0 . 05 ( grip ) ; * P < 0 . 05 ( body weight ) ; * P < 0 . 05 ( rotarod ) ; and * P < 0 . 05 ( survival ) . ( E ) Representative photograph of a 13 - week - old AR - 97Q mouse treated with DMSO ( left ) and an age - matched AR - 97Q mouse treated with 2 . 0 mg / dL RG108 ( right ) . AR - 97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO - treated AR - 97Q mouse of same age . The white compact masses ( arrow ) of mice head are the cement covering the surgical site . ( F , G ) Immunoblots ( F ) and relative intensities ( G ) of the Dnmt1 - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) . ( H , I ) Dnmt1 immunohistochemistry in spinal cord sections from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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Figure 3(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).


Figure 4 ( A ) Immunohistochemistry of the spinal cords of AR - 97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody . ( B ) Quantification of 1C2 - positive motor neurons in the spinal cords ( n = 5 per group ) . ( C ) Immunoblotting of the spinal cords of AR - 97Q mice treated with or without RG108 for AR . ( D ) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR - 97Q mice treated with DMSO or RG108 ( n = 3 ) . ( E ) Relative mRNA expression levels of human AR in AR - 97Q mice spinal cord with or without RG108 treatment ( n = 3 ) . Immunohistochemistry for ChAT in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) . quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) . ( H , I ) Western blot analysis ( H ) and relative signal intensities ( I ) of the ChAT - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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Figure 4(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).


Figure 5 ( A ) Immunoblotting of SH - SY5Y cells stably expressing AR - 24Q ( SH24Q ) or AR - 97Q ( SH97Q ) for Dnmt1 , Dnmt3a and Dnmt3b . ( B ) Densitometry quantification showing that Dnmt1 level was up - regulated in SH97Q cells compared with SH24Q cells ( n = 3 ) . ( C , D ) Immunoblots for Dnmts of SH97Q with or without DHT treatment ( C ) and quantification data ( D ) ( n = 3 ) . ( E ) Seven genes with DNA hyper - methylation in their CpG islands were identified by DNA methylation array analysis . ( F ) The relative mRNA levels of CDC25B , GFRA3 , NPY , HES5 , SCTR , LEF1 - AS1 and CABS1 , normalized to beta - 2 microglobulin , in DHT - treated SH24Q and SH97Q cells were measured using RT - qPCR ( n = 3 ) . ( G ) RT - qPCR analysis indicated the decrease of Hes5 mRNA level in DHT - treated SH97Q cells compared with DHT - untreated SH97Q cells ( n = 3 ) . ( H ) Methylation - specific PCR of SH24Q and SH97Q cells that were treated with DHT . ( I ) Hes5 immunoreactivity of spinal motor neurons in wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) . ( J ) Hes5 mRNA levels in the spinal cords of wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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Figure 5(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b. (B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).(J) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).


Figure 6DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108 . HES5 mRNA levels of SH97Q cells treated with DHT and RG108 . DNA methylation levels of the Hes5 promoter region in DHT - treated NSC97Q cells treated with or without RG108 . mRNA levels of Hes5 ( n = 3 ) in DHT - treated NSC97Q cells treated with or without RG108 . ( E ) Hes5 mRNA levels in the spinal cords of AR - 97Q mice treated with or without RG108 ( n = 3 ) . ( F , G ) Hes5 immunoreactivity of spinal motor neurons in AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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Figure 6DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.HES5 mRNA levels of SH97Q cells treated with DHT and RG108.DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).


Figure 7 ( A ) Viability of DHT - treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST - 8 assay ( n = 3 ) . ( B ) Immunoblots of DHT - administrated NSC97Q cells treated with Hes5 siRNA ( n = 3 ) . ( C ) Quantitative cell viability analysis revealed that siRNA - mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT ( n = 3 ) , as determined by the WST - 8 assay . Cell viability measured with the WST - 8 assay of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) . Cell viability measured with immunoblotting of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) . ( F ) Quantification of LDH release from DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 4 ) . ( G ) Immunoblots of DHT - administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR . ( H ) AR aggregation as quantified by densitometry ( n = 3 ) .

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Figure 7(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).


Figure 8 ( A ) Immunoblots of NSC34 cells treated with Hes5 siRNA . ( B ) Quantification of protein levels of pSmad2 , Smad2 , phospho - IkBα and heat shock factor - 1 ( Hsf1 ) ( n = 3 ) . ( C ) siRNA - mediated knockdown of Hes5 down - regulated Hes5 protein level ( n = 3 ) . ( D ) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment ( n = 3 ) . ( E ) Protein levels of Smad2 , pSmad2 , and Hes5 in NSC97Q cells transfected with Hes5 vector ( n = 3 ) . ( F ) Immunoblots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q ( n = 3 ) . ( G ) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q ( n = 3 ) . ( H ) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q ( n = 6 ) . ( I ) Western blots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5 - containing lentiviral vector .

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 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 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 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 B-PANEL_START 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 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 8(A) Immunoblots of NSC34 cells treated with Hes5 siRNA. (B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).(E) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.


Figure 1 ( B ) Structured illumination microscopy image of perimCherry / cytoGFP E . coli confirming localization of mCherry ( red ) in the periplasm and GFP ( green ) in the cytosol . Scale bar = 3 µm . ( C ) Outer membrane damage ( mCherry intensity ) and inner membrane damage ( % Sytox positive ) of perimCherry / cytoGFP E . coli bacteria exposed to ( different concentrations of ) human serum . Inner membrane damage correlates with killing ( samples where bacteria are killed are indicated with gray shadings and a cross , see CFU data in Fig EV1b ) . Data information : ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D ) Serum - induced inner membrane damage ( % Sytox positive ) of different Gram - negative strains depends on MAC components C5 and C8 , but not on lysozyme ( 10 % serum ) . Dotted line represents the detection limit of the assay . Data information : ( D , E ) The cfu / ml ( Fig . 1E , 2C ) and sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " and " C5b6MAC " were all generated from the same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 . , E Serum - induced killing ( CFU / ml ) of different Gram - negative strains depends on MAC components C5 and C8 , but not on lysozyme ( 10 % serum ) . Dotted line represents the detection limit of the assay . Data information : ( D , E ) The cfu / ml ( Fig . 1E , 2C ) and sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " and " C5b6MAC " were all generated from the same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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 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 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

Figure 1(B) Structured illumination microscopy image of perimCherry/cytoGFP E. coli confirming localization of mCherry (red) in the periplasm and GFP (green) in the cytosol. Scale bar = 3 µm.(C) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments.(D) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001., E Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


Figure 2 ( B ) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7 . After washing , erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured . ( B - E ) Data represent mean ± SD of 3 independent experiments . C ) Bacterial viability of three Gram - negative strains after exposure to buffer , 10 % human serum or C5b6MAC . Buffer and serum conditions are the same as Fig . 1E . ( D ) Permeabilization of the outer , but not inner membrane of perimCherry / cytoGFP E . coli cells exposed to C5b6MAC ( different concentrations of C5b6 with fixed concentrations of C7 - C9 ) . ( B - E ) Data represent mean ± SD of 3 independent experiments . E ) Inner membrane damage of three Gram - negative strains exposed to buffer , 10 % serum or C5b6MAC . Buffer and serum conditions are the same as Fig . 1D .

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 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 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

Figure 2(B) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7. After washing, erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured. (B-E) Data represent mean ± SD of 3 independent experiments.C) Bacterial viability of three Gram-negative strains after exposure to buffer, 10% human serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1E.(D) Permeabilization of the outer, but not inner membrane of perimCherry/cytoGFP E. coli cells exposed to C5b6MAC (different concentrations of C5b6 with fixed concentrations of C7-C9). (B-E) Data represent mean ± SD of 3 independent experiments.E) Inner membrane damage of three Gram-negative strains exposed to buffer, 10% serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1D.


Figure 3 ( B ) Bacterial viability of convertase - labeled bacterial strains exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 . ( C ) Bacterial viability of convertase - labeled E . coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6 , 100 nM C7 , 20 nM C8 and 100 nM C9 . ' Ctrl ' indicates bacteria that are pretreated with heat - inactivated ΔC5 serum . Dotted line represents the detection limit of the assay . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( D ) Bacterial viability of convertase labeled E . coli MG1655 exposed to C5 - C9 or conditions lacking one MAC component . As an extra control , convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 . ( E ) Bacterial viability of E . coli MG1655 exposed to FB depleted serum in the presence of 20 µg / ml OmCI ( to deposit C4b and C3b without Bb ) . After washing , bacteria were exposed to C5 - C9 in the presence or absence of C1 and C2 ( to generate classical pathway C5 convertases , C4b2aC3b ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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 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 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 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

Figure 3(B) Bacterial viability of convertase-labeled bacterial strains exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.(C) Bacterial viability of convertase-labeled E. coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6, 100 nM C7, 20 nM C8 and 100 nM C9. 'Ctrl' indicates bacteria that are pretreated with heat-inactivated ΔC5 serum. Dotted line represents the detection limit of the assay. Data information: (B-E) Data represent mean ± SD of 3 independent experiments.(D) Bacterial viability of convertase labeled E. coli MG1655 exposed to C5-C9 or conditions lacking one MAC component. As an extra control, convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin. Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.(E) Bacterial viability of E. coli MG1655 exposed to FB depleted serum in the presence of 20 µg/ml OmCI (to deposit C4b and C3b without Bb). After washing, bacteria were exposed to C5-C9 in the presence or absence of C1 and C2 (to generate classical pathway C5 convertases, C4b2aC3b). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


Figure 4 ( A ) Outer membrane damage ( mCherry intensity ) and inner membrane damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli cells incubated with a concentration range of C5 and fixed concentrations of C6 - C9 . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( B ) Inner membrane damage of perimCherry / cytoGFP E . coli exposed to a concentration range of ΔC5 serum and , after washing , to C5 - C9 . As controls , bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( C ) Inner membrane damage of three different convertase - labeled bacteria exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( C ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 or * * p ≤ 0 . 01 . ( D ) Confocal microscopy images of convertase - labeled perimCherry / cytoGFP E . coli exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Unlabeled bacteria exposed to 1 % serum served as control . Green = GFP , Red = To - pro - 3 DNA dye . Scale bars = 3 µm .

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 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 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

Figure 4(A) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.(B) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.(C) Inner membrane damage of three different convertase-labeled bacteria exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05 or ** p≤0.01.(D) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.


Figure 5 ( B ) Bacterial viability of convertase - labeled E . coli MG1655 exposed to Buffer ( Conv ) , preassembled C5b6 ( Conv + C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of C7 , C8 and C9 . Dotted line represents the detection limit of the assay . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 . ( C ) Inner membrane damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 . ( D ) outer membrane damage ( mCherry ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 .

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 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 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

Figure 5(B) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.(C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.(D) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


Figure 6A , B Step - wise assembly of MAC on convertase - labeled bacteria . Convertase - labeled bacteria were incubated with C5 / C6 or C5 / C6 / C7 for 15 minutes , and subsequently washed ( @ ) or treated with 10 μg / ml Eculizumab ( Ecu ) . Then , the remaining MAC components ( C7 - 9 for C5 / C6 or C8 - 9 for C5 / C6 / C7 respectively ) were added to the incubation mixture . In the control conditions ( Conv - MAC ) , the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor . ( A ) Outer membrane ( mCherry ) and ( B ) inner membrane damage ( % Sytox positive ) were determined . Data information : ( A - B ) Data represent mean ± SD of 3 independent experiments . Statistical analysis was done using a ratio paired two - tailed t - test in which the test conditions were compared to Conv - MAC and displayed only when significant as * p ≤ 0 . 05 .

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

Figure 6A, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.


Figure 7 ( A ) Outer and inner membrane damage of convertase - labeled bacteria exposed to different combinations of MAC components . ' @ ' indicates a washing step . Data information : Data represent mean ± SD of 3 independent experiments . ( A ) Statistical analysis was done using a one - way ANOVA and displayed only when significant as * * p ≤ 0 . 01 or * * * * p ≤ 0 . 0001 . ( B ) Outer and inner membrane damage of convertase - labeled bacteria exposed to C5 - C8 and after washing , to a concentration range of C9 . Data information : Data represent mean ± SD of 3 independent experiments .

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 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

Figure 7(A) Outer and inner membrane damage of convertase-labeled bacteria exposed to different combinations of MAC components. '@' indicates a washing step. Data information: Data represent mean ± SD of 3 independent experiments. (A) Statistical analysis was done using a one-way ANOVA and displayed only when significant as ** p≤0.01 or **** p≤0.0001.(B) Outer and inner membrane damage of convertase-labeled bacteria exposed to C5-C8 and after washing, to a concentration range of C9. Data information: Data represent mean ± SD of 3 independent experiments.