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.