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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Nat Med. 2015 Oct 12;21(11):1307–1317. doi: 10.1038/nm.3960

Figure 4. aPC-EPCR-PAR1 signaling retains HSCs by inducing Cdc42 polarity and stabilizing VLA4.

Figure 4

(a) Cdc42 (purple) distribution in bone marrow EPCR+ (yellow) and EPCR− Lineage negative Sca-1+ (pink) c-Kit+ (red) cells. Scale bar, 7 µm; n = 4. (b) Cdc42 (green) distribution in bone marrow EPCR+ (yellow) SK SLAM cells pretreated with EPCR non-inhibitory or EPCR neutralizing antibody. Scale bar, 7 µm; n = 5. (c) Active Cdc42-GTP (green) in bone marrow EPCR+ SK SLAM (yellow) and EPCR− LSK cells. Scale bar, 7 µm; n = 5. (d) Active Cdc42-GTP (green) in bone marrow EPCR+ (yellow) SK SLAM cells pretreated with EPCR non-inhibitory or EPCR neutralizing antibody. Scale bar, 7 µm; n = 4. (e) VLA4 expression on bone marrow EPCR+ or EPCR− SK SLAM cells; n = 4. (f) VLA4 affinity measured by LDV probe binding to bone marrow EPCR+ SK SLAM cells following in vitro stimulation with aPC of cells that had been pretreated with EPCR neutralizing antibody or control EPCR non-inhibitory antibody for 30 minutes; n = 7. (g) VLA4 affinity of bone marrow SK SLAM cells from wild-type or Procrlow mice following in vitro treatment with aPC for 1 hour; n = 3. (h) VLA4 expression on bone marrow SK SLAM cells from wild-type or Procrlow mice; n = 3. (i) VLA4 expression on bone marrow SK SLAM cells and VLA4 affinity on bone marrow EPCR+ SK SLAM cells from wild-type or F2r−/− mice; n = 3. (j) Peripheral blood LSK and EPCR+ LSK cell frequencies following EPCR or VLA4 neutralization in vivo; n = 4, P values, one-way ANOVA with Tukey’s post-test for left panel and Dunn’s post-test for right panel.