Figure 4.

NRP1 Regulates Iron Homeostasis And Iron-Dependent Oxidative Stress

(A) Intracellular total iron (Fe²⁺ plus Fe³⁺) of HDMECs si-control or si-NRP1 expressed as percentage relative to si-control (mean ± SEM; n = 3).

(B) HDMECs si-NRP1 or si-control were incubated with 5 μM Mito-FerroGreen (gray) and 300 nM MitoTracker Deep Red FM (red); scale bar, 20 μm.

(C) Mito-FerroGreen fluorescent signal per cell was quantified and expressed as percentage relative to si-control (mean ± SEM; n = 3).

(D) HDEMCs si-NRP1 or si-control were stained for ferroportin-1 (green or gray) and counterstained with DAPI (blue); scale bar: 20 μm.

(E) Integrated density of ferroportin-1 was normalized to DAPI and expressed as fold change of si-control (mean ± SEM; n = 4).

(F) HDMECs treated with deferoxamine 100 μM for 24 hr after 48 hr from transfection with si-NRP1 or si-control were incubated with 100 nM TMRM (gray); scale bars, 20 μm.

(G) Integrated density of the TMRM signal normalized to cell number determined by manual counting using high-contrast images (panel F, green images) and expressed as percentage relative to si-control (mean ± SEM; n = 4).

(H) HDMECs si-control or siNRP1 untreated or treated with deferoxamine 100 μM for 24 hr were incubated with 5 μM MitoSOX (red), counterstained with Hoescht 33342 (blue); scale bar: 20 μm.

(I) Integrated density of the MitoSOX signal expressed as percentage relative to untreated si-control (mean ± SEM; n = 4).

*p < 0.05, ***p < 0.001; Student's t test. See also Figure S3.