Figure 5: None

(A) Reverse transcription quantitative–polymerase chain reaction of representative genes after PLAGL1 or STAT1 knockdown in aHCFs. Whereas common human cardiac fibroblast genes VIM and SMA are unchanged, genes associated with the adult phenotype are down-regulated (ELN and TNFRSF11B), and HBEGF (associated with fHCFs) is up-regulated. Apoptosis-related genes such as BAX, CASP3, PCNA, and MCM3 are in particular up-regulated after STAT1 knockdown. Similarly, cellular senescence marker MMP3, as well as RB1 and TP53 showed strong upregulation following STAT1 knockdown. Collagen genes (COL1A1 and COL1A2) are down-regulated upon knockdown of both TFs. Values are mean ± SD and taken from at least n = 3. All p < 0.05, Student t test. Biological repeats from independent aHCF cell isolates are shown in Supplemental Figure 6. (B) Apoptosis was assessed by using the ApopTag-Red assay that detects DNA fragmentation. A low number of cells (6%) exhibited apoptosis under control conditions (control Neg. A GapmeR transfection), and this was significantly up-regulated in the positive control (DNAse I–treated cells) as well as after PLAGL1 and STAT1 knockdown. A strikingly high level of cytoplasmic ApopTag-labeling was repeatedly evident in STAT1 knockdown cells, which was not seen in others. (C) Heterochromatin foci were detected in 9% (450 of 5,595 nuclei analyzed) of STAT1 knockdown cells. These foci frequently, but not exclusively, overlapped with the ApopTag-Red stain. (D) Nuclei illustrating that heterochromatin foci were never found in Ki67-positive cells. Abbreviations as in Figures 1, 3, and 4.