Figure 4. Extracellular sAPPα or CP increases cellular expression of surface ferroportin.

FPN location was examined in (A) HEK293T and (B - D) primary murine neuronal cultures, preincubated with iron (50 µM, 3 h) followed with CP or sAPPα (1 µM, 30 min). Both cell lines have been previously shown to utilize APP to promote iron efflux, and do not express CP [2], [14]. Surface proteins on (A) HEK293T cells, and (B) primary neurons, were biotinylated to identify changes to endogenous FPN and APP expression on the cell surface, as well as exogenously attached sAPPα or CP. Surface levels of FPN were significantly increased in the presence of CP or sAPPα, despite total levels of FPN remaining unchanged. The graphs show the distribution of FPN when normalized against the β-actin content of the intracellular+surface fractions, and adjusted for protein load. Similar results for FPN distribution were achieved even without adjusting for β-actin (not shown). (C) Fluorescence-activated cell sorting of non-permeabilized N2a neuroblastoma cultures preincubated with iron (50 µM, 6 h) confirms an increase in surface expression of FPN, quantified in (D), after a 30 min incubation with sAPPα (1 µM) in OptiMEM. (E) Deconvoluted confocal microscopy shows overlap of endogenous APP and FPN at the surface of non-permeabilized primary neurons preincubated with iron (50 µM, 3 h), as well as (F) increased FPN on the neuronal surface following further treatment with CP or sAPPα (1 µM, 30 min). Endogenous surface FPN was below detection limits in neurons that were not treated with FAS (not shown). Data in (A), (B) & (D) are means ± S.E. of 3 experiments, performed in duplicate. * p<0.05, ** p<0.01 and *** p<0.001 analyzed treatment vs control, by two-tailed t tests. (C) is a representative histograms of>10,000 live cells normalized to the control (treated with secondary antibody only) mean signal, set at 10². (E) & (F) are representative images of a neuron from 2 experiments, performed in duplicate. Scale bar = 10 µm.