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. 2019 Sep 25;9(24):7282–7297. doi: 10.7150/thno.33920

Figure 4.

Figure 4

Safe and Sfrp2 mutually regulates each other's RNA stability in fibroblasts. (A) Schematic diagram of RNA-RNA interaction between Safe and Sfrp2 at their reverse complementary regions (462 nucleotides in length). Thick blocks and thin blocks represent the coding sequence (CDS) and untranslated regions (UTRs), respectively. (B) qRT-PCR detection of Sfrp2 expression in left ventricles of sham and MI mice at indicated days after surgery (n=3). (C) qRT-PCR detection of Sfrp2 expression in the infarct zone (IZ), border zone (BZ), and remote zone (RZ) of infarcted hearts after 14 days following MI surgery (n=3). (D) qRT-PCR detection of Sfrp2 expression in aCM, aCF and aEC (n=3). (E) qRT-PCR analysis showing increased expression of Sfrp2 in cardiac fibroblasts after TGF-β treatment (n=3). (F) mRNA expression of Sfrp2, Col1a1, α-SMA and Bmp1 in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Sfrp2 knockdown (n=3). (G) Representative western blot analysis and relative densitometric quantification of SFRP2, COL1A1 and α-SMA protein levels in TGF-β-untreated or TGF-β-treated cardiac fibroblasts after Sfrp2 knockdown (n=3). (H) qRT-PCR analysis showing decreased expression of both Safe and Sfrp2 mRNA in cardiac fibroblasts transfected with shSafe (n=3). (I) Representative western blot analysis and relative densitometric quantification of SFRP2 protein with Safe inhibition (n=)3. (J) qRT-PCR analysis showing decreased expression of both Sfrp2 mRNA and Safe in cardiac fibroblasts transfected with shSfrp2 (n=3). (K) Subcellular localization of Sfrp2 in cytoplasm and nuclei of cardiac fibroblasts (n=3). (L) qRT-PCR analysis showing preferentially inhibition of Sfrp2 mRNA level in the nucleus of cardiac fibroblasts after Safe knockdown (n=3). (M) Representative images of RNA FISH showing co-localization of Safe (green), Sfrp2 mRNA (magenta) in the nuclei (DAPI, blue) of cardiac fibroblasts. Scale bar: 20 μm. (N) Dual luciferase assay showing shSafe inhibited Firefly luciferase activities of pGL3-control vectors carrying Sfrp2 3'-UTR (n=3). (O) Dual luciferase assay showing shSfrp2 inhibited Firefly luciferase activities of pGL3-control vectors carrying Safe RNA fragments complementary to Sfrp2 mRNAs (n=3). All data are presented as mean ± SEM; Student's t-test or one-way ANOVA; *p < 0.05, and ns, not significant.