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Figure 3 | Arthritis Research & Therapy

Figure 3

From: Interleukin-17A promotes functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells by extracellular-regulated protein kinases signalling pathway

Figure 3

IL-17A induces proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs via ERK1/2 signing pathway. (A) DVSMCs were pre-treated with PD 98059 (10 μM/ml) for 2 h before incubation with IL-17A at 100 ng/ml for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were pre-treated with PD98059 for 2 h before incubation with the serum of SSc patients or healthy individuals for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A at 100 ng/ml for 24 h. The activity of collagen1α1 or collagen3α1 proximal promoter was detected using a dual luciferase reporter gene assay. (D) After being pre-treated with PD98059 for 2h before incubation with the serum of SSc patients or healthy individuals for 24 h, the activity of collagen1α1 or collagen3α1 proximal promoter was also detected. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E, F) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G, H) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (I, J) The cells were pre-treated with PD98059 for 2 h before incubation with IL-17A, serum from SSc patients and healthy individuals for 24 h, and the protein expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

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