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. Author manuscript; available in PMC: 2020 Jan 29.
Published in final edited form as: Circulation. 2019 Jan 29;139(5):679–693. doi: 10.1161/CIRCULATIONAHA.118.034615

Figure 4. LMO7 inhibits AP-1 activity and TGF-β1 autoinduction.

Figure 4

(A) qPCR analysis of Jun and Fos genes in medial layers of freshly isolated mouse aortas (n=4 independent experiments). (B) Mouse SMCs were treated with 0.5ng/ml TGF-β1 for 24hrs and mRNA was harvested for qPCR analysis of Jun and Fos genes (n=5 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and TGF-β1 treatment on mRNA expression but there was no significant interaction between Lmo7−/− and TGF-β1 treatment. (C) Western analysis of human CASMCs transfected with control or LMO7 siRNA for 72 hrs. Quantification of c-JUN and c-FOS is shown on right (n=4 independent experiments). (D) Chromatin immunoprecipitation (ChIP) with c-JUN or c-FOS antibodies from human CASMCs transfected with control or LMO7 siRNA for 72 hrs, followed by qPCR analysis using primers flanking the AP-1 binding sites in TGFB1 or ITGB3 promoters (n=4 independent experiments). (E, F) Lmo7+/+ or Lmo7−/− mice were subjected to carotid ligation and injected intratracheally with T5224 (30mg/kg/d) or Vehicle (polyvinylpyrrolidone solution) daily from day 4 post-ligation. Mice were sacrificed at day 14 and carotid arteries were harvested. Trichrome (E) or p-SMAD3 (F) staining of cross-sections of injured arteries is shown. p-SMAD3 staining intensity per cell area were measured and shown on right (n=4). Two-way ANOVA revealed a significant effect of LMO7 depletion and T5224 treatment on p-SMAD3 staining intensity and the magnitude of reduction by T5224 treatment was greater in Lmo7−/− mice (p=0.0013), indicating LMO7 regulates TGF-β activity by modulating AP-1 activity. (G, H) Mouse SMCs were treated with 40 μM T5224 for 24hrs and cell lysates were harvested for (G) western analysis of SMAD3 phosphorylation (n=3 independent experiments) or (H) qPCR analysis of Tgfb1, Itgav, Itgb3, Jun and Fos genes (n=3 independent experiments) Hprt1 was used as a negative control in qPCR analysis. Two-way ANOVA revealed a significant effect of LMO7 depletion and T5224 treatment on p-SMAD3 phosphorylation and mRNA expression and the magnitude of reduction by T5224 treatment was greater in Lmo7−/− SMCs for Itgb3 (p=0.026) and Fos (p=0.019) mRNA only. (I) Lmo7+/+ or Lmo7−/− mouse SMCs with the same confluency were treated with 40 μM T5224 or Vehicle for 24 hrs. Conditioned media was harvested to quantitate total TGF-β secretion by ELISA (n=3 independent experiments). Two-way ANOVA revealed a significant effect of LMO7 depletion and T5224 treatment on TGF-β1 production, but there was no significant interaction between Lmo7−/− and T5224 treatment. Data are expressed as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001