Fig. 3

In vitro anti-angiogenic capacity of Lf-GL. a Relative viability of HUVEC cells treated with GL equivalent concentration of 25 μM to 200 μM, respectively, in GL-, Lf-, and Lf-GL- treated group. Data are expressed as mean ± S.E.M (n = 6). ***P < 0.001 versus GL-treated group. b Monitoring endothelial cell proliferation for 3 days in GL-, Lf-, and Lf-GL- treated HUVEC. Data are expressed as mean ± S.E.M (n = 6). ***P < 0.001 versus GL-treated group. c Investigation of HMGB1-mediated HUVEC proliferation and inhibitory ability of GL, Lf, and Lf-GL for 3 days. Data are expressed as mean ± S.E.M (n = 8). d Capillary tube formation assay to investigate HMGB1-mediated angiogenesis and its inhibitory effect of GL, Lf, and Lf-GL. Scale bar: 200 μm. e Quantification of tube formation in the absence or presence of HMGB1 in control, GL, Lf, and Lf-GL groups. HMGB1 (-) indicates absence of HMGB1 treatment. HMGB1 ( +) indicates the presence of HMGB1 treatment. Data are expressed as mean ± S.E.M (n = 3). *P < 0.05 and ***P < 0.001. f Western blot analysis for phospho- and total ERK levels in cell lysates. g Densitometric analysis of the related bands was expressed as the relative optical band density, which was corrected using total ERK proteins as a loading control and normalized against the untreated control. HMGB1 (-) indicates the absence of HMGB1 treatment. HMGB1 ( +) indicates the presence of HMGB1 treatment. Data are expressed as mean ± S.E.M (n = 3). *P < 0.05 and **P < 0.01. h Rat aortic ring assay to evaluate anti-angiogenic effect of GL, Lf, and Lf-GL under condition where endothelial cells, progenitor cells, and other angiogenic factors are interacting. Scale bar: 200 μm. i Quantification of micro-vessel sprouting. Data are expressed as mean ± S.E.M (n = 6). *P < 0.05