Fig 3.
Lamstatin and CP17 inhibit cord formation and migration of human lung lymphatic endothelial cells (HMVEC-LLys) in vitro. HMVEC-LLys were seeded on Matrigel (BD Bioscience) for 1 hr to allow attachment before EBM-2 MV medium (complete) with increasing concentration of lamstatin, CP17 or vehicle was added. Cord formation was assessed after 24 hrs. (A) Cord formation images from a representative experiment. Lamstatin (10 μg/ml) compared with vehicle control (1 nM EDTA, pH 3.5) and CP17 (10 μg/ml) compared with vehicle control (sterile MilliQ water). Black scale bar represents 500 μm. (B) Cord formation in the presence of lamstatin (white bars) (n = 3 experimental repeats) or CP17 (black bars) (n = 3 experimental repeats). Data are expressed as mean ± SE of the mean (SEM) of cords per 1000 square pixel (px2) and one-way repeated measures anova for comparisons of various concentrations versus no treatment, *P < 0.05. (C) HMVEC-LLys were seeded at 4.0 × 105 cells/ml per transwell and serum starved (EBM-2 MV, no supplements, 0.1% BSA) before EBM-2 MV medium (complete) with lamstatin (white bars) (n = 3 experimental repeats) or CP17 (black bars) (n = 3 experimental repeats) was added. Cells were allowed to migrate for 24 hrs and then stained with Calcein-AM for detection. Data were normalized to vehicle controls and presented as mean ± SEM per cent migration (100% VC: vehicle control). One-way repeated measures anova treatment versus no treatment, *P < 0.05.