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. 2026 Apr 30;29(6):115958. doi: 10.1016/j.isci.2026.115958

Figure 4.

Figure 4

BVRA oxidoreductase activity is dispensable for heme-induced NRF2 activation and insulin receptor signaling in BMDM

(A) BMDM from BlvraWT, Blvra−/−, BlvraG17A, and BlvraE97A mice were stimulated with heme and NRF2 activation assessed by immunofluorescence.

(B) Representative immunofluorescence images of NRF2 nuclear translocation in BMDM following heme stimulation. Images show confocal z stack cross-sections with orthogonal views. NRF2 is shown in red, DAPI nuclear stain in blue, and phalloidin (cytoskeleton marker) in white. Scale bars, 5 μm.

(C) Quantification of cytoplasmic (left graph) or nuclear (right graph) NRF2 MFI in BMDM stimulated with (+) or without (−) heme. Each data point represents a technical replicate. n = 5–10 replicates. Data in (C) is representative from two independent experiments with a similar trend.

(D) BMDM from BlvraWT, Blvra−/−, BlvraG17A, and BlvraE97A mice were stimulated with heme and NRF2-regulated gene expression assessed by qRT-PCR.

(E) RT-qPCR analysis of NRF2 target genes (Nqo1, Fth, Gclc, Hmox1, Nrf2, and Blvra) in BMDM stimulated with (+) or without (−) heme. Data expressed as gene/Rplp0 (2−ΔCt). Each data point represents a technical replicate. n = 3–6 replicates from two independent experiments with a similar trend.

(F) BMDM from BlvraWT, Blvra−/−, BlvraG17A, and BlvraE97A mice were stimulated with insulin and INSR signaling assessed by western blot.

(G) Representative western blots showing IRS-1 phosphorylation at tyrosine 612 (IRS-1Y612), at serine 307 (IRS-1S307), total IRS-1, and vinculin as loading control in BMDM stimulated with insulin vs. control vehicle.

(H) Quantification of IRS-1Y612 normalized to IRS-1S307 in BMDM stimulated with (+) or without (−) insulin. Each data point represents a technical replicate. n = 3 replicates from one experiment. Data in (C, E, and H) are presented as individual data points with bar graphs showing mean ± SD. Statistical comparisons were performed using two-way ANOVA with Šídák’s post hoc test. ∗p < 0.05, ∗∗p < 0.01; NS, not significant.