Figure 3
(A) Flow cytometric analysis of clathrin-mediated endocytosis of fluorescently labelled transferrin (Alexa 488) in BeWo cells. Cells were pre-cultured (48 h) with 2 mM (n = 14) or 2.5 mM (n = 21) glucosamine (Gln), before the addition of transferrin (6.25ug/ml) for 15 min at 37 °C (± Chlorpromazine, CPMZ clathrin inhibitor). Internalised transferrin is displayed as mean fluorescence per event in 10,000 events, as a fold change from the positive control (indicated by the green intercepting line). (B) Assessment of clathrin-mediated endocytosis of fluorescently labelled transferrin in first trimester human placental tissue (8.5–12 weeks gestation). Tissue explants cultured (48 h) with glucosamine ex vivo were further cultures in serum depleted medium with glucosamine (3 h) to measure the rate of uptake of transferrin (50 μg/ml). Following culture tissues were acid washed (20 s) and lysed (RIPA buffer). Fluorescent intensity of lysate read on a plate reader and normalised to total protein concentration. −ve: negative control tissue was not exposed to transferrin. Data displayed as mean and SEM, Wilcoxon signed ranked statistical analysis was used, where *p = 0.05, **p = 0.01, ***p = < 0.001 and ****p = < 0.0001 versus positive control.
