Fig. 7: Effects of protein glycosylation inhibitors on BCEC proliferation.
a Dose-dependent stimulation of BCEC proliferation by various inhibitors of glycosylation. Inhibitors were added at concentrations ranging from 0.01 to 100 μM for 3 days, with or without 5 ng/ml VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. A representative experiment is shown. Kifunesine (Kif), an ERα-1,2-mannosidase I and Golgi α-mannosidase I inhibitor; Castanospermine (Cas), an a-glucosidase inhibitor. Cell-covered areas in various treatment groups were quantified by ImageJ software. b Dose-dependent effects of Kif and Cas in promoting BCEC proliferation with or without 5 ng/ml VEGF. n = 3 independent samples. c Both inhibitors reduced VEGFR2 molecular mass and induced Bip expression in a dose-dependent fashion as assessed by western blot analysis. Proteins from total cell lysates were separated using 3–8% Tris-Acetate gel. BCECs were treated with various inhibitors for 24 h. Quantification of western blots was done by densitometry. β-actin was the loading control. d Acceleration of closure of monolayer gaps by Kif and Cas in BCEC scratch assay, with controls for Kif (H2O) and Cas (DMSO). Gaps were quantified using AxioVision LE Rel.4.4 software. n = 3 independent samples. Scale bar = 400 µm. e Activation of AKT and JNK in BCECs by glycosylation inhibitors at 40 μM and VEGF at 10 ng/ml. However, Cas did not activate ERK. Quantification of phosphorylated AKT, JNK, and ERK was done by densitometry analysis relative to total protein. f Pre-treatment of BCECs with 5 μM SP600125 for 2 h significantly blocked the effects of both glycosylation inhibitors on BCEC proliferation. n = 3 independent samples. A representative experiment is shown from two to four independent studies. Data shown are means +/− SD. Statistical analysis was done by two-tailed, two-sample unequal variance t test. *p < 0.05, **p < 0.01. Data are provided as a Source data file.
