Fig. 1: BM ECs upregulate expression of SEMA3A and its receptor, NRP1, following irradiation.

a Heatmap of semaphorin (Sema) gene expression in irradiated and non-irradiated (non-IRR) BM ECs isolated before and after irradiation (n = 3/condition). b qRT-PCR analysis of Sema and Nrp expression in VE-cad⁺ BM ECs from Non-IRR and 500 cGy-irradiated mice (n = 6/condition from n = 10,000 cells/replicate; ****p < 0.0001). Data normalized to non-IRR sample. Horizontal bars represent means. c qRT-PCR of Sema3a expression in BM hematopoietic cell populations, LepR⁺ stromal cells and CD31⁺Sca-1⁻ sinusoidal BM ECs (sBMECs) and CD31⁺Sca-1⁺ arterial BM ECs (aBMECs; n = 4–12 replicates/condition; ****p < 0.0001). Data normalized to KSL cells. d qRT-PCR of Sema3a expression in aBMECs and sBMECs before and +24 h following 500 cGy (n = 6/group; aBMEC: ***p = 0.0008, sBMEC: n.s. p = 0.72). Data normalized to non-IRR. e qRT-PCR of Nrp1 expression in BM hematopoietic cell populations, LepR⁺ stromal cells and ECs (n = 4–12 replicates/group; ****p < 0.0001). Data normalized to KSL cells. f SEMA3A concentration in the BM of mice following 500 cGy quantified by ELISA (n = 8 mice; 6 h, d1, d2: ****p < 0.0001, d7: ***p = 0.0001, d14: ***p = 0.0007). Dotted line represents BM SEMA3A concentration in non-IRR mice. g Confocal images of femurs from C57BL/6J mice before and +72 h following 500 cGy. Expression of SEMA3A (yellow) in VE-Cad⁺ BM ECs (magenta) and CD31⁺ BM ECS (green) is shown (white arrows). Nuclei labeled with DAPI (blue). Representative images from n = 3 experiments, Scale bar 50 µm; magnified scale bar, 20 µm; max z-projections shown. h Expression of NRP1 (yellow) in VE-Cad⁺ BM ECs (magenta) and CD31⁺ BM ECS (green) is shown (white arrows) in same conditions. Representative images from n = 3 experiments. i Representative gating, histogram, and j quantification of NRP1 expression on Non-IRR and 500 cGy-irradiated BM ECs (n = 5/group, ****p < 0.0001, aBMEC: p = 0.8256). k NRP1 MFI within BM EC sub-populations before and +24 h post-500 cGy (n = 5/group, ****p < 0.0001). Data assessed by Holm-Sidak’s multiple comparison two-sided t-test after two-way ANOVA (b, d, f, j, k) and the Holm-Sidak’s multiple comparison two-sided t-test after one-way ANOVA (c, e). Data presented as mean values +/− SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file. .