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. Author manuscript; available in PMC: 2018 Sep 27.
Published in final edited form as: Neuron. 2017 Sep 27;96(1):115–129.e5. doi: 10.1016/j.neuron.2017.09.003

Figure 6. ApoE4 Impairs IR Trafficking by Trapping IR in the Endosome. See also Figure S8 and S9.

Figure 6

SH-SY5Y neuronal cells transfected with human IR-GFP were treated overnight with recombinant apoE3 or apoE4 (100 nM) in the presence or absence insulin (100 nM) stimulation for 30 minutes. Alexa Flour 568-labeled human transferrin (Tf, 20 μg/ml) or Lysotracker (10 μM) was added to the medium one hour before fixing the cells and images were obtained by confocal microscopy. (A) The co-localization of IR-GFP (Green) and Tf (Red) in apoE3- and apoE4-treated cells in the presence and absence of insulin is shown. DAPI (blue) was included to position the nucleus. (B) The co-localization of IR-GFP (Green) and Lysotracker (Red) in apoE3- and apoE4-treated cells in the presence and absence of insulin is shown. DAPI (blue) was included to position the nucleus. (C) Percentage of intracellular IR (intra-IR/total IR) was calculated. (D) Percentage of IR that was co-localized with Tf (Tf co-localized IR) was calculated. (E) Percentage of IR that was co-localized with Lysotracker (Lysotracker co-localized IR) was calculated. Data from three independent experiments are expressed as mean ± SEM. Two-tailed student’s t test was used for statistical analysis. ***p < 0.001; ****p < 0.0001; N.S., not significant. Scale bar, 20 μm.