Fig 1.
Evaluation of CD68, CD204 and CD206 expression in cultured THP-1-derived macrophages.
Immunocytochemistry of CD68 (marker of macrophage activation), CD204 and CD206 protein expression in cultured THP-1-derived macrophages (M0 macrophages) treated for 72 hours with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured M0 macrophages maintained for 72 hours in RPMI at 5% of FBS were used as controls (M0-controls). Immunocytochemistry was performed on four independent in vitro experiments. The detection of protein expression was performed evaluating the same number of cells by light microscopy (magnification 40X).
Fig 2.
Evaluation of CD68, CD204 and CD206 expression in cultured human monocyte-derived macrophages.
Immunocytochemistry of CD68 (marker of macrophage activation), CD204 and CD206 protein expression in cultured human monocyte-derived macrophages treated for 6 days with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured human monocyte-derived macrophages maintained for 6 days in RPMI at 10% of FBS were used as untreated cells. Immunocytochemistry was performed on four independent in vitro experiments. The detection of protein expression was performed evaluating the same number of cells by light microscopy (magnification 40X).
Fig 3.
Evaluation of protein synthesis of M2 phenotype markers in cultured THP-1-derived macrophages.
Western blotting and related densitometric analysis of CD204, CD206 and CD163 protein synthesis in cultured THP-1-derived macrophages (M0 macrophages) treated for 72 hours with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured M0 macrophages maintained for 72 hours in RPMI at 5% of FBS were used as controls (M0-controls). Western blotting was performed on six independent in vitro experiments. The data of CD204, CD206 and CD163 protein synthesis are shown as mean±SD and indicated as increase in protein synthesis.
Fig 4.
Evaluation of protein synthesis of M2 phenotype markers and TGFbeta1 in cultured human monocyte-derived macrophages.
Western blotting and related densitometric analysis of CD204, CD206, CD163 and TGFbeta1 protein synthesis in cultured human monocyte-derived macrophages treated for 6 days with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured human monocyte-derived macrophages maintained for 6 days in RPMI at 10% of FBS were used as untreated cells. Western blotting was performed on six independent in vitro experiments. The data of CD204, CD206, CD163 and TGFbeta1 protein synthesis are shown as mean±SD and indicated as increase in protein synthesis.
Fig 5.
Evaluation of gene expression of M2 macrophage phenotype markers in cultured THP-1-derived macrophages.
Quantitative real time polymerase chain reaction (qRT-PCR) of CD204, CD206, CD163, IL-10 and CCL-22 gene expression in cultured THP-1-derived macrophages (M0 macrophages) treated for 72 hours with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured M0 macrophages maintained for 72 hours in RPMI at 5% of FBS were used as controls (M0-controls). The qRT-PCR was performed on six independent in vitro experiments and the data of CD204, CD206, CD163, IL-10 and CCL-22 gene expression are shown as mean±SD and indicated as increase in gene expression.
Fig 6.
Evaluation of gene expression of M2 macrophage phenotype markers and TGFbeta1 in cultured human monocyte-derived macrophages.
Quantitative real time polymerase chain reaction (qRT-PCR) of CD204, CD206, CD163, IL-10, CCL-22 and TGFbeta1 gene expression in cultured human monocyte-derived macrophages treated for 6 days with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before being stimulated with ET-1. Cultured human monocyte-derived macrophages maintained for 6 days in RPMI at 10% of FBS were used as untreated cells. The qRT-PCR was performed on six independent in vitro experiments and the data of CD204, CD206, CD163, IL-10, CCL-22 and TGFbeta1 gene expression are shown as mean±SD and indicated as increase in gene expression.
Fig 7.
Evaluation of pro-fibrotic MMP-9 production in cultured macrophages.
Evaluation by gel zymography of MMP-9 production and related densitometric analysis in cultured M0-macrophages and human monocyte-derived macrophages. (A) Cultured M0 macrophages were treated for 72 hours with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (bosentan, 10-5M) for 1 hour before the stimulation with ET-1. Cultured M0 macrophages maintained for 72 hours in RPMI at 5% of FBS were used as controls (M0-controls). (B) Cultured human monocyte-derived macrophages treated for 6 days with ET-1 (100nM) and IL-4 (10ng/mL) alone, or pre-treated with ETA/BRA (10-5M) for 1 hour before the stimulation with ET-1. Cultured human monocyte-derived macrophages maintained for 6 days in RPMI at 10% of FBS were used as untreated cells. Gel zymography was performed on six independent in vitro experiments and the data of MMP-9 production are shown as mean±SD.