Figure 5.

NRP1 Regulates Iron-Dependent Oxidative Stress via ABCB8

(A) Quantification of ABCB8 mRNA by RT-qPCR in HDMECs si-ABCB8, si-NRP1, or si-control. Transcripts levels were expressed as fold change of si-control (mean ± SEM; n = 3).

(B) Immunostaining for ABCB8 (green) in HDMECs si-ABCB8 or si-control counterstained with DAPI (blue); scale bar, 20 μm.

(C) ABCB8 integrated density in optical z stacks was normalized to DAPI integrated density and expressed as percentage relative to si-control (mean ± SEM; n = 3).

(D) HDMECs treated with deferoxamine 100 μM for 24 hr, 48 hr after transfection with si-ABCB8 or si-control were incubated with 100 nM TMRM (gray); scale bar, 20 μm.

(E) Integrated density of the TMRM signal was normalized to cell number determined by manual counting using high-contrast images (Figure 5D, green panels) and expressed as percentage (mean ± SEM; n = 4).

(F) HDMECs si-ABCB8 or si-control were incubated with 5 μM MitoSOX (red) and counterstained with Hoescht 33342 (blue); n = 4; scale bar, 20 μm.

(G) Integrated density of the MitoSOX signal was quantified as percentage of si-control (mean ± SEM; n = 4).

(H) Quantification of NRP1 and ABCB8 mRNA by RT-qPCR in HDMECs single or double transfected with si-NRP1 and si-ABCB8 or si-control (mean ± SEM; n = 3).

(I) HDMECs single or double transfected with si-ABCB8 and si-NRP1 or si-control were incubated with 100 nM TMRM (gray); scale bar, 20 μm.

(J) Integrated density of the TMRM signal was normalized to cell number determined by manual counting using high-contrast images (panel I, green images) and expressed as percentage (mean ± SEM; n = 3).

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