Fig 1: In vitro knockdown of core cardiac transcription factors.(a) Images of adult cardiac fibroblasts derived from ROSACas9-GFP mice, 24 and 72 hr post-transfection with two guide RNAs for GFP and CleanCap mCherry mRNA. (b) Flow cytometry plot showing the expression on mCherry in 67% of transfected cells (representative image of three independent experiments). (c) Relative quantification qPCR of GFP, normalized by Gapdh expression, in cells transfected with scrambled or GFP guide RNAs. (d, e) Volcano plots showing genes differentially expressed in Gata4 (left) and Tbx20 wns (right). Fold change in expression of Gata4 (left) and Tbx20 (bottom) in cells transfected with specific gRNAs versus scramble RNA, quantified through RNA sequencing. (f) Venn diagram showing the overlap among genes affected by Gata4 (Gata4KD) or Tbx20 (Tbx20KD) knockdown and number of genes upregulated by tenfold or more in heart fibroblasts (eHF) compared to other organs. (g) Plot showing changes in expression of eHF genes affected by Gata4 (in blue) or Tbx20 (in red) knockdown. (h) Genes regulated by both Gata4 and Tbx20 in opposing manner. (i) KEGG pathway analyses. Top panel: 58 genes affected in both Tbx20 and Gata4 knockdown; middle panel: 594 genes affected by Gata4 knockdown; bottom panel: 66 genes affected by Tbx20 knockdown. Blue – pathway changed in opposite directions, red – upregulated pathways, and green – downregulated pathways. (j) Hand-picked genes illustrate alterations in processes known to affect the cardiac fibrotic response for Gata4 or Tbx20 knockdown. All data are represented as fold changes over scrambled control (average ± standard error of the mean [SEM]; d–j) from bulk RNAseq of three biological replicates per condition. Selected significant genes have an FDR < 0.05. Refer to Figure 5—source data 1. Figure 5—source data 1.CRISPR-Cas9 experiments: sequence of the guide RNAs; differentially expressed genes between Tbx20KD and Gata4 KD and corresponding controls.KEGG pathways analysis.