Fig 4. Acute deletion of Robo4 did not alter BM VECs or sinusoidal VCAM1 expression.

A. Representative flow cytometry plots for the BM VEC and SEC populations. VECs were defined as CD45^-/Ter119^-/CD31⁺/Sca1⁺ BM cells. Ai. SECs were additionally defined as Tie2⁺ and Sca^mid and evaluated for VCAM1 expression. B. Germline, but not acute, deletion of Robo4 resulted in increased numbers of BM VECs. The germline R4^KO mice exhibited significantly more VECs compared to Wt mice, whereas neither the pan- (R4^cKO by Rosa26-CreERT2) nor vascular-specific (R4^ECcKO by VEcad-CreERT2) acute Robo4 deletion exhibited significantly different numbers of total VECs. We have previously published similar results for the R4^KO mice [17]; this panel includes both previously reported and new data from the R4^KO performed side-by-side with the two conditional models. Data represent n = 4–6 independent experiments with n = 37 wt, n = 37 R4^KO, n = 18 tamoxifen treated Wt, n = 15 R4^cKO, and n = 10 R4^ECcKO. Statistics by unpaired two tailed student’s t-test. ** p < 0.01. C. Unlike the germline R4^KO, the frequency of VCAM1 on sinusoidal BM VECs was not significantly different upon acute pan or endothelial-specific deletion of Robo4. R4^KO mice exhibited significantly lower frequency of VCAM1⁺ sinusoidal VECs compared to Wt cohorts, whereas R4^cKO or R4^ECcKO did not. We have previously published similar results for the R4^KO mice; this panel includes both previously reported and new data from the R4^KO performed side-by-side with the two conditional models [17]. Data represent n = 4 independent experiments, n = 21 Wt, n = 21 R4^KO, n = 18 tamoxifen treated Wt, n = 15 R4^cKO, and n = 10 R4^ECcKO. Statistics by unpaired two tailed student’s t-test. * p < 0.05. See also S2 Fig. E. Unlike the germline R4^KO, the number of BM SECs (CD45^-/Ter119^-/Sca1^low/Tie2⁺) was not significantly different upon acute pan or endothelial-specific deletion of Robo4. R4^KO mice exhibited significantly lower BM sinusoidal VECs compared to Wt cohorts, whereas R4^cKO or R4^ECcKO did not. We have previously published similar results for the R4^KO mice; this panel includes both previously reported [17] and new data from the R4^KO performed side-by-side with the two conditional models. Data represent n = 4 independent experiments, n = 21 Wt, n = 21 R4^KO, n = 18 tamoxifen treated Wt, n = 15 R4^cKO, and n = 10 R4^ECcKO. Statistics by unpaired two tailed student’s t-test. * p < 0.05.