Fig. 3

EC miR-126 downregulation associates with CD31⁺Sca-1^high EC depletion in the BM niche. a Representative plots (left) and aggregate results (right) of Sca-1^low frequency in cultured Sca-1^high ECs upon miR-126 knock-down (KD) by GFP-expressing miRZip anti-miR-126 lentivirus transduction, as analyzed by flow cytometry (n = 3). b Representative plots (left) and aggregate results (right) of Sca-1^high frequency in cultured Sca-1^low ECs upon miR-126 over-expression (OE) by GFP-expressing miR-126 precursor lentivirus transduction, as analyzed by flow cytometry (n = 3). c miR-126 levels in ECs from the BM of wt, miR-126^ECΔ/Δ (EC miR-126 KO) and Spred1^ECΔ/Δ (EC miR-126 OE) mice by Q-RT-PCR (n = 4 mice per group). d and e CD31 (FITC) and Sca-1 (PE) IF staining (d) and quantification (e) of CD31⁺Sca-1^high EC-lined vessels (i.e., arterioles) in the tibias from wt, miR-126^ECΔ/Δ and Spred1^ECΔ/Δ mice. One of the three independent experiments with similar results is shown. Yellow arrows indicate CD31⁺Sca-1^high EC-lined vessels; white arrows indicate CD31⁺Sca-1^low EC-lined vessels. f and g Representative plots (f) and aggregate results (g) of Sca-1^high and Sca-1^low subfractions in BM ECs from wt, miR-126^ECΔ/Δ and Spred1^ECΔ/Δ mice by flow cytometry analysis (n = 3 mice per group). h CD31 (FITC) and Sca-1 (PE) IF staining (top) and quantification (bottom) of CD31⁺Sca-1^high EC-lined vessels (i.e., arterioles) in the tibias from normal wt mice treated with CpG-scramble (SCR) or CpG-miR-126 inhibitor (miRisten, 20 mg/kg/day, iv, for 3 weeks; n = 3 mice per group). Yellow arrows indicate CD31⁺Sca-1^high EC lined-vessels; white arrows indicate CD31⁺Sca-1^low EC-lined vessels. i Representative plots (left) and aggregate results (right) of Sca-1^high and Sca-1^low subfractions in BM ECs from normal wt mice treated with SCR or miRisten for 3 weeks (n = 3 mice per group). Results represent mean ± SEM. Significance values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001