Figure 3.

Inhibition of clathrin mediated endocytosis by romidepsin. (a) Effects of XJY drugs on cellular uptake of transferrin. Hela cells were treated with indicated concentrations (0, 50, 500 μM) of drugs, followed by incubation with fluorescently labeled Alexa-555 transferrin (red) for 15 min at 37 °C. Cells were then washed with fresh medium and fixed for microscopy imaging. Scale bar is 20 μm. (b) Concentration-dependent inhibitory effect of romidepsin on transferrin internalization was as shown. (c) Time-dependent inhibitory effect of romidepsin on transferrin internalization. Cells were preincubated with indicated time before incubation with Alexa-555 transferrin. Cells were harvested and analyzed as in panel a; 10 μM romidepsin was used in this experiment. (d,e), Prolonged treatment can lower effective dose of romidepsin for inhibition transferrin uptake. Hela cells pretreated with 2 or 12 h of romidepsin were incubated with transferrin. The internalization of transferrin was respectively examined at 5, 10, and 15 min after the addition of transferrin. For quantification of the endocytosis, the total intracellular transferrin-555 signal was measured from 50 cells in each experimental condition.