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. Author manuscript; available in PMC: 2018 Apr 19.
Published in final edited form as: Handb Exp Pharmacol. 2018;245:271–312. doi: 10.1007/164_2017_62

Figure 6. Clathrin-dependent and independent endocytotic pathways regulate hA internalization in pancreatic cells.

Figure 6

(A) hA monomer internalization is independent of clathrin and dynamin at 1 hour in RINm5F cells. Cells were treated with dynamin inhibitor dynasore (Dyn) or clathrin inhibitor chlorpromazine (Chl) for 1 hour followed by hA (green) (10 μM) for an additional 1 hour at 37°C. In parallel, cells were incubated with hA (10 μM) for 1 hour at 4°C. CTX (red) (20 μg/ml) and Trf (blue) (50 μg/ml) were finally added for 30 minutes at 37°C or 4°C. Whole cell analysis (graph) demonstrated no noticeable difference in cellular distributions of monomers at 1 hour when treated with Dyn or Chl. However, lowering temperature to 4°C blocked monomer internalization as well as CTX and Trf (A). Arrowheads and arrows denote cells with internalized and PM associated hA monomers, respectively. (B) Late phase of hA monomer internalization requires clathrin in RINm5F cells. Cells were first transfected with wild type (wt-AP180) or dominant negative clathrin adaptor AP180 protein, containing clathrin binding domain at its C-terminus (DN AP180CFLAG) for 16–18 hours. Following transfections, cells were incubated with 10 μM hA (green) for an additional 24 hours at 37°C. Cells were also treated with hA at the indicated concentrations for 24 hours at 4°C. CTX (red) (20 μg/ml) and Trf (blue) (50 μg/ml) were finally added for 30 minutes at 37°C or 4°C after incubating the cells with hA. Confocal microcopy and whole cell analysis revealed a significant reduction in internalization and an increase in PM accumulation of hA when transfected with DN AP180CFLAG or when incubated at 4°C. In contrast, there was no change in their cellular distributions in wt-AP180 expressed cells and controls (B). Transferrin but cholera toxin internalization was blocked in cells transfected with DN AP180CFLAG construct (B). Internalization of all three cargoes were effectively blocked at 4°C (B). **p<0.01, hA vs. hA plus inhibitors, ##p<0.01, hA vs. hA/4°C, ##p<0.01, CTX vs. CTX/4°C, and Trf vs. Trf/wt-AP180, NS p>0.05, n=9. Significance established by ANOVA followed by Dunnett-Square test. Bar 10μm.