Inhibition of RUNX1 attenuates the E-selectin expression. (A) Relative expression of E-selectin, P-selectin, VCAM1, TIE2, SDF1 (CXCL12), ICAM1, and JAG1 was determined in HUVECs treated with control DMSO or Chb-M′ at 0.5 μM or 1.0 μM. Twenty-four hours after treatment, total RNA was prepared and processed for real-time qPCR analysis. Values are normalized to those of DMSO-treated cells (n = 3). (B) Correlation between the expression levels of RUNX1 and E-selectin in HUVEC cell lines established from 9 different umbilical cords (n = 9). Values represent array signal intensities. P values were determined using Spearman’s correlation. (C) Surface expressions of E-selectin were determined in HUVEC treated either by control DMSO or Chb-M′ at 1 μM. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (D) Relative expression of E-selectin, P-selectin, VCAM1, TIE2, SDF1 (CXCL12), ICAM1, and JAG1 was determined in HUVECs transduced with control (sh_Luc.) or RUNX1 shRNA (sh_RUNX1) in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and processed for real-time RT-PCR analysis. Values are normalized to that of control cells (n = 3). (E) Surface expression of E-selectin was determined in HUVECs transduced with sh_Luc. or sh_RUNX1 in the presence of 3 μM doxycycline. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (F) Schematic illustration showing the proximal regulatory region (−600 bp to +200 bp of the transcription start site) of E-selectin. (G) ChIP analysis in HUVECs using anti-RUNX1 antibody, an isotope-matched control IgG, and anti-histone H3 antibody. ChIP products were subjected to PCR-based amplification with the indicated primer sets (supplemental Table 1), using RPL30 as a negative control. (H) Schematic representation of the treatment and analysis schedule in C57BL/6 mice. Mice were treated with andrographolide (25 mg/kg body weight, 3 times/week, IP), A 205804 (10 mg/kg body weight, 3 times/week, orally [p.o.]), or Chb-M′ (320 μg/kg body weight, 3 times/week, IV) for 2 weeks. After treatment, mice were sacrificed, and endosteal cells were dislodged from the femurs for further analysis. (I) Representative flow cytometry analysis identifying vascular niche cells in the endosteal cells obtained in panel H. Expression of E-selectin was determined in the indicated endothelial cells (Lin⁻CD45⁻CD31⁺). (J) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or andrographolide. (K) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or A 205804. (L) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or Chb-M′. Data are presented as mean ± SEM. *P < .05, **P < .01 (2-tailed Student t test). FSC, forward scatter; SSC, side scatter.