Co-treatment with interferon-γ and 1-methyl tryptophan ameliorates cardiac fibrosis through cardiac myofibroblasts apoptosis

Fig. 1. Growth suppression of hCMs by IFN-γ. a Cell viability of hCMs treated with IFN-γ. hCMs were treated with 100 ng/ml IFN-γ. Cell viability was evaluated by performing MTT assays. Data are expressed as the mean ± SE of triplicate experiments. **P < 0.01 b Cell cycle analysis of hCMs treated with IFN-γ for 72 h. Cellular DNA contents were analyzed by flow cytometry. The data shown are from one of three independent experiments. *P < 0.05

Fig. 2. Signaling pathways activated by IFN-γ in hCMs. a hCMs were treated with IFN-γ (100 ng/ml) for the indicated time points, and the expression of proteins related to apoptosis (i.e., IRF-1, Fas, and FasL) or cell cycle arrest (i.e., IDO1 and IDO2) was detected by immunoblotting. In addition, STAT1 phosphorylation and α-SMA expression were also evaluated. *P < 0.05 Black arrows: IDO bands; red arrows: non-specific (ns) bands. b Kynurenine production in hCMs treated with IFN-γ for 72 h. The data shown are expressed as the mean ± SE of triplicate experiments. ***P < 0.001. (Color figure online)

Fig. 3. Release from IFN-γ-induced cell cycle arrest by inhibiting IDO activity. a, b Growth-inhibitory effects of IFN-γ (100 ng/ml) and/or 1-MT (0.5 mM) in hCMs. hCMs were treated with IFN-γ or IFN-γ + 1-MT for the indicated time points, and cell viability was examined by light microscopy (a) or MTT assays (b). *P < 0.05, **P < 0.01, and ***P < 0.001 c Kynurenine production in hCMs treated with IFN-γ or IFN-γ + 1-MT for 48 or 72 h. The data shown are expressed as the mean ± SE of triplicate experiments. ***P < 0.001

Fig. 4. Cell death induced by IFN-γ and 1-MT in hCMs. a Cell death of hCMs induced by IFN-γ and 1-MT. hCMs treated with IFN-γ + 1-MT for 72 h were analyzed by PE-annexin-V and 7-AAD staining, and cell death was quantified by flow cytometry. Necrotic (PE-annexin-V/7-AAD, ±), late apoptotic (PE-annexin-V/7-AAD, +/+), live (PE-annexin-V/7-AAD, –/–), and early-apoptotic cells (PE-Annexin-V/7-AAD, ±) are shown in Q1–Q4, respectively. b Change in the cell cycle phase induced by IFN-γ + 1-MT. Cellular DNA contents were analyzed by flow cytometry. The data shown are expressed as the mean ± SE of triplicate experiments. *P < 0.05 and **P < 0.01 c Signaling pathways activated by IFN-γ and 1-MT in hCMs. In hCMs treated with IFN-γ (100 ng/ml) and 1-MT (0.5 mM) for the indicated time points, the expression of apoptosis-related proteins (i.e., IRF-1, Fas, and FasL) were analyzed by immunoblotting. The y axis of the lower left graph is the logarithmic scale. *P < 0.05 and **P < 0.01. Additionally, p-STAT1, α-SMA, IDO1, and IDO2 were also detected. Black arrows: IDO bands; red arrows: non-specific (ns) bands. (Color figure online)