Fig. 2. Role of RNA in cfB production in the heart and isolated cardiomyocytes.

A. RNA digestion by serum RNase - Testing the efficacy of systemic administration of RNase. As detailed in the Methods, sera were prepared from mice injected with saline or RNase A, and incubated with 3 μg of purified RNA at 37°C for 2 hours. Lane 1: Untreated RNA; Lane 3–8: RNA treated with serum from the mice injected with saline; Lane 9–14: RNA treated with serum from the mice injected with RNase A. B–D. Effect of systemic RNase administration on cardiac gene expression of cfB, C3 and C5. RNase was administrated before and 12h after sham or CLP surgery. cfB/C3/C5 gene expression was detected in the heart 24h after surgery using qRT-PCR. n=4 in each group. # P < 0.05, δ P < 0.001 vs. Sham-Saline; ** P < 0.01. E. Effect of RNase on RNA-induced cfB protein production. Cultured rat neonatal cardiomyocytes were treated lipofectamine alone (Lipo), or with cardiac RNA (10 μg/ml) for 24 hours. As indicated, in some treatment groups, RNA was first pretreated with RNase or DNase before applied to cardiomyocyte cultures. Medium cfB was detected by Western blot as described in the Methods. F. Effect of TLR ligands on cfB protein production in rat neonatal cardiomyocytes. Cardiomyocytes were treated with poly (I:C) (10 μg/ml), R837 (1 μg/ml), or CL075 (1 μg/ml) for 24 hours. Medium cfB was detected with Western blot.