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. 2017 Jun 1;7(8):2186–2203. doi: 10.7150/thno.18407

Figure 2.

Figure 2

IL-32α inhibits vascular smooth muscle cell (VSMC) migration and proliferation. VSMCs were isolated from thoracic aortas of hIL-32α-Tg and non-Tg mice. (A) VSMCs were cultured in an ibidi μ-Dish with culture inserts for 24 h. Cells were then treated with PDGF-BB (25 ng/mL; 24 h) in serum-free media, and cell migration was monitored for 18 h by taking pictures every 3 h. The area of migrated cells was quantified (n = 3). Images shown are representative microscopy images at 18 h. (B) VSMCs were cultured in 96-well plates for 24 h, serum-starved for 12 h, then treated with PDGF-BB (25 ng/mL; 24 h), and cell proliferation was determined by BrdU incorporation assay (n = 5 each). (C-D) VSMCs from non-Tg mice were treated with rhIL-32α (25-100 ng/mL) for 24 h in serum-free medium, then cells were treated with PDGF-BB (25 ng/mL; 24 h). (C) cell migration (n = 3) and (D) cell proliferation (n = 5) were determined as described above (data shown as mean ± SEM, *p < 0.05 by Student's t-test). Images shown in C are representative microscopy images for each group. (E) VSMCs were lysed and analyzed by western blotting with antibodies against PCNA, p21, phospho-ERK1/2, and total-ERK1/2, using β-actin as a loading control. (F) For in vitro MMP activity assays, cells were cultured in 96-well plates for 24 h, then treated with PDGF-BB (25 ng/mL; 24 h). MMP activity was determined by cell-based enzyme-linked immunosorbent assay (ELISA) using DQ™-gelatin (data shown as mean ± SEM; n = 8, *p < 0.05 as determined by Student's t-test). (G) To evaluate specific MMPs activity, cells were cultured in 6-well plates for 24 h, then treated with PDGF-BB (25 ng/mL; 24 h), then gelatinolytic activity (MMP-2; 72 kDa) was determined in culture medium using gelatin zymography followed by Coomassie blue staining. GM6001 (40 μM), a broad-spectrum MMP inhibitor, was used as negative control. Bar diagrams presented fold change of MMP-2 considering the serum-free control density as 1 (data shown as mean ± SEM, n = 3, *p < 0.05 as determined by Student's t-test).