Blood, Vol. 114, Issue 17, 3557-3566, October 22, 2009

Loss of the Rho GTPase activating protein p190-B enhances hematopoietic stem cell engraftment potential
Blood Xu et al. 114: 3557

Supplemental materials for: Xu et al

Bone marrow collection and lineage depletion
BM cells were flushed from femora and tibiae and suspended in Hanks balanced salt solution (HBSS; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Atlanta Biologicals, Laurenceville, GA), and 1% penicillin (10000 U/mL)/streptomycin (5000 µg/mL; Cambrex Bio Science, East Rutherford, NJ). Mononuclear cells were isolated by low-density centrifugation (Histopaque 1083; Sigma-Aldrich, St Louis, MO) and incubated with a cocktail of biotinylated antibodies against epitopes on differentiated leukocytes: anti-CD11b (clone M1/70), anti-B220 (clone RA3-6B2), anti-CD5 (clone 53-7.3), anti–Gr-1 (clone RB6-8C5), anti-Ter119, anti-CD8a (Clone 53-6.7; all from BD Pharmingen, San Diego, CA). Antibody-labeled Lin+ cells were removed using sheep anti-rat IgG magnetic immunobeads (Dynal Inc., Lake Success, NY, USA).

Immunodetection of proteins
Lysates from fetal liver cells were prepared in ice-cold lysis buffer containing protease inhibitors. Debris was removed by centrifugation in a microfuge at 12,000 × g for 10 min at 4°C. Clarified lysates were boiled in gel loading buffer and separated by SDS-PAGE. Proteins were electrotransferred to nitrocellulose and detected with antibodies directed against p190-A RhoGAP (BD Transduction Laborarories, Lexington, KY), p190-B RhoGAP (Cell Signaling Technology Inc., Beverly, MA).

RhoGTPase activity assays
Lineage-depleted cells were lysed with Mg2+-based lysis buffer and assessed for Rac and Cdc42 pull down assay using PAK-PBD (Upsate, Charlottesville, VI) or for RhoA using Rhotekin-PBD, as previously described.20 Total cell lysates were analyzed for Cdc42, Rac or RhoA expression with anti-Cdc42 or anti-Rac (Transduction Laboratories, San Diego, CA), or anti-RhoA (Santa cruz) as loading control.

Files in this Data Supplement:

  • Table S1. Blood counts of reconstituted mice (PDF, 14.2 KB)

  • Table S2. Lineage distribution of donor-derived cells in peripheral blood and BM during serial transplantation (PDF, 65 KB)

  • Figure S1. WT and p190-B−/− (JPG, 197 KB) -
    (A) Expression of p190-B and p190-A in WT and p190-B−/− fetal liver cells analyzed by immunoblot. (B) Activity of RhoA, Cdc42, and Rac in WT and p190-B−/− lineage-depleted cells assessed by the pull down assay using the PAK-binding domain for Cdc42 and Rac or the Rhotekin-binding domain for RhoA. The numbers represent the relative ratio of GTP-Rho GTPase versus total protein.





  • Figure S2. p190-B−/− FL cells are able to reconstitute the hematopoiesis in lethally irradiated mice (JPG, 328 KB) -
    (A) Reconstitution of FL cells. 2 × 106 total FL cells from E14.5 embryos were injected into lethally irradiated Ly5.2 mice. Histogram represents the percentage of donor-derived cells in the PB at the indicated time points after transplantation (mean ± SD, n=5). (B & C) Lineage reconstitution in PB and BM for reconstituted mice. Donor contribution to B (B220), T (CD3ε) and myeloid (Mac-1 and Gr-1) cells were analyzed by FACS on gated CD45.2+ PB (B) and BM (C) cells at 4 months post transplantation. Histogram represents the percentage of each lineage in donor-derived cells (mean ± SD, n=5). (D) BM cellularity of reconstituted mice at 4 months post transplantation. Histogram represents the BM cellularity in reconstituted mice as contained in 2 femora, 2 tibiae and pelvis (mean ± SD, n=5). (E) Frequency of LSKCD150+CD48−, LSKCD34−, LSK and LK cells in the BM of reconstituted mice. BM cells from reconstituted mice at 4 months post transplantation were stained with anti-CD45.2, c-Kit, Sca-1, CD34, and a cocktail of lineage Abs, CD34− cells in the LSK fraction were detected by FACS. Histogram represents the percentage of LSKCD34− , LSK cells and LK cells (mean ± SD, n=5).