Fig 1: Ccn3 abundance decreases with depletion of Yap/Wwtr1. (A) Western blots depicting protein abundance of Yap, Wwtr1, or Ccn3 following siRNA treatment in cultured neonatal rat cardiac fibroblasts with Gapdh loading controls. (B) Quantification of Ccn3 protein from neonatal rat cardiac fibroblasts following siRNA, normalized to non-treated samples. n = 3 biological replicates per group. Each replicate consisted of cells from independent litters. One-way ANOVA comparing experimental groups to negative siRNA control by Dunnett's test. (C) Western blot depicting Ccn3 abundance in left ventricles of uninjured and 14 dpi left ventricles. (D) Feature plot illustrating the abundance of Ccn3 expressing cells by UMI within clusters 5 and 16. Ns, not significant and *P < 0.05.
Fig 2: Ccn3 administration promotes fibrotic gene expression post MI. (A) Experimental timeline of echocardiography, MIs, CCN3 administration, and EdU administration in adult mice. (B) Quantification of left ventricular function by fractional shortening, ejection fraction, and left ventricular internal diameters during diastole and systole at baseline. 3, 14, and 28 dpi timepoints were analyzed by repeated measures two-way ANOVA and Sidak multiple comparisons test. n = 10 PBS treated, 9 CCN3 treated. (C) Quantification of heart and body weights at 28 dpi. n = 10 PBS treated, 9 CCN3 treated. Unpaired student's t-test. (D) Representative serial sections of Gömöri trichrome stained hearts measured as distance from the apex. Scale = 5 mm. (E) Quantification of infarct scar size by either total fibrotic area or midline size of the left ventricle. n = 10 PBS treated, 9 CCN3 treated. Unpaired student's t-test. (F) Representative immunohistological images denoting EdU+ nuclei within the scar area. White arrows indicate EdU+ scar associated nuclei. Scale = 100 µm. (G) Quantification of EdU+ scar associated nuclei. n = 7 PBS treated, 6 CCN3 treated. Scale bar = 100 µm. Unpaired student's t-test. (H) Representative images and (I) quantification of WGA staining in the remote zone of the left ventricle. n = 9 PBS treated, 9 CCN3 treated. Scale = 100 µm. Unpaired student's t-test. ns, not significant. F-H Are from 28 dpi hearts. ns, not significant, *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001.
Fig 3: Ccn3 administration promotes fibrotic gene expression post MI. (A) PCA of DEGs from RNAseq from left ventricles. n = 3 per group. (B) Heatmap of 309 genes with one-way ANOVA FDR P-value <0.2. The top three canonical pathways identified for each gene cluster by IPA are listed. (C,D) The top cellular component terms derived from upregulated (red) or downregulated (blue) DEGs derived from DAVID analysis. (F) The top upstream regulators from all DEGs predicted by IPA. (G) The top upregulated and downregulated genes by fold-change mediated by Tead1 or Tgfβ1.
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