Figure 3. Fibroblast-specific deletion of Klf5 in Klf5^fl/fl;Postn-Cre mice.

(A) Fibroblast-specific deletion of the floxed region in Postn-Cre mice was examined using R26RstoplacZ indicator mice. LacZ expression was visualized using X-gal. Scale bars: 100 μm. (B) CD3^– cells within non-myocyte-enriched cell populations isolated from adult hearts were analyzed for surface expression of the fibroblast marker Thy1 and the endothelial marker CD31. (C) Relative expression levels of cell-lineage markers in adult cardiomyocytes (CM) isolated using the Langendorff perfusion method, and in Thy1⁺CD31^–CD3^– (Thy1⁺) and Thy1^–CD31⁺CD3^– (CD31⁺) cells sorted from non-myocyte-enriched populations as shown in B. Myh6 (encoding αMHC), Ddr2 (encoding discoidin domain receptor 2), and Cdh5 (encoding VE-cadherin) were used as markers for cardiomyocytes, fibroblasts, and ECs, respectively. The cells were isolated from 8-week-old mice subjected to sham operations. (D) Competitive PCR analysis for quantitation of Cre-mediated recombination of the Klf5 gene region in adult cardiomyocytes, CD31⁺ ECs, and Thy1⁺ fibroblasts isolated from Klf5^fl/fl and Klf5^fl/fl;Postn-Cre mice 2 weeks after either the sham or LI-TAC operation. Competitive PCR was performed as shown in Supplemental Figure 6B. (E) Relative expression levels of Klf5 mRNA in adult cardiomyocytes, Thy1⁺ fibroblasts, and CD31⁺ ECs isolated from Klf5^fl/fl and Klf5^fl/fl;Postn-Cre mice as shown in B 5 days after either sham operation or LI-TAC. Expression levels of Klf5 mRNA were assessed using real-time PCR and normalized to those of 18s rRNA, after which they were further normalized to the levels in Thy1⁺ cells isolated from Klf5^fl/fl mice subjected to the sham operation. *P < 0.01 versus sham control of the same genotype in the same cell lineage group; ^#P < 0.01 versus Klf5^fl/fl mice subjected to LI-TAC in the same cell lineage group.