Xavier-Ferrucio J , Ricon L , Vieira K , Longhini AL , Lazarini M , Bigarella CL , Franchi G , Krause DS , Saad STO .

R01 DK086267 NIDDK NIH HHS , R01 DK094934 NIDDK NIH HHS , R01 DK114031 NIDDK NIH HHS , U54 DK106857 NIDDK NIH HHS

	Fig. 2. Arhgap21 deficiency in progenitor cells leads to increased mobilization and defective adhesion. (A–D) Hematopoietic cells were analyzed by flow cytometry 1 h after AMD3100 treatment. (A) Bone marrow from Arhgap21+/− mice exhibited a reduction of Gr-1+/Mac-1+ cells compared to WT (P = 0.001), whereas the frequencies of Gr-1+/Mac-1+ cells in spleen (P = 0.02) and peripheral blood (P = 0.007) were increased. (B) The change in the frequency of Gr-1+Mac-1+ cells before and after AMD3100 for each genotype is shown (after AMD3100 treatment as shown in Fig. 2A) minus before AMD3100 treatment as in Fig. 1J). (C) The frequency of LSK cells was not significantly altered in the bone marrow and spleen, whereas the frequency of LSK was increased in peripheral blood (P = 0.0005) of Arhgap21+/− mice after AMD3100 treatment (WT n = 7 and Arhgap21+/− n = 7). (D) The change in the frequency of LSK cells before and after AMD3100 for each genotype is shown ((after AMD3100 treatment as shown in Fig. 2C) minus before AMD treatment as in Fig. 1B). (E) Adhesion to fibronectin was impaired in Lin− cells obtained from Arhgap21+/− mice (WT n = 5 and Arhgap21+/− n = 5; P = 0.003). (F) Arhgap21+/− mice showed a reduction of Lin− cells expressing CD49d/CD29 (a4b1 integrin) in bone marrow (P = 0.04), but no difference in the frequency of Lin− cells expressing CD49e/CD29 (a5b1 integrin). (G) Similarly, the mean fluorescence intensity of α4β1 integrin in Lin− Arhgap21+/− cells were also decreased (P = 0.02) when compared to WT.
	Fig. 3. Impaired function of Arhgap21+/− hematopoietic stem and progenitor cells in vitro and in vivo. (A-C) Short-term reconstitution was evaluated by hematopoietic recovery after 5-FU treatment. (A) We observed a higher frequency of Lin−Sca+cKit+ (LSK) cells 28 days after 5-FU treatment (P = 0.04). (B–C) Peripheral blood was collected 7, 14, 21 and 28 days after treatment and blood cell count was performed by automated hematology analyzer. (B) There is an increase in the neutrophil number of Arhgap21 mice 14 (P = 0.0005) and 21(P = 0.0002) days after 5-FU treatment. (C) Despite an increase in the LSK frequency, there is a decrease in erythroid reconstitution 7 (P = 0.03) and 14 days (P < 0.0001) after the 5-FU treatment. (D-E) Bone marrow cells from wild type and Arhgap21+/− mice were cultured in methylcellulose media supplemented with cytokines for myeloid differentiation and the total number of colonies was scored after ten days of incubation. (D) Colonies were then replated in methylcellulose and secondary CFU formation was accessed after an additional 10 days (Primary: P = 0.007, Secondary: P = 0.02). (E) When analyzed by frequency, there is a decrease of BFU-E percentage in the haploinsufficient mice (P = 0.03), suggesting that the erythroid compartment is the most affected by the reduction of Arhgap21. (F-G) For CFU-S, bone marrow cells were injected into lethally irradiated WT mice, and spleen colonies counted after 12 days. (F) Macroscopic view of CFU-S in recipients that received cells from WT and Arhgap21+/− mice. (G) Quantification of CFU-S colonies showing reduced number of CFU-S in mice that received Arhgap21+/− cells (P = 0.002). (H–I) Serial bone marrow transplantation was performed using bone marrow cells from Arhgap21+/− and WT mice transplanted into lethally irradiated CD45.1 mice. Chimerism (CD45.2+) in the peripheral blood of the recipients was examined at indicated times after primary and secondary transplantation, showing a slight reduction of donor cells in PB of Arhgap21+/− mice, 4 weeks after primary transplant (P = 0.04). (I) Donor derived (CD45.2+) immature LSK cells, which were analyzed in bone marrow 16 weeks after primary and secondary transplantation by flow cytometry, are reduced in recipient mice that received Arhgap21+/− bone marrow in secondary transplantation (P = 0.001). Minimum of 8 WT and 8 Arhgap21+/− animals used in each experiment.
	Fig. 4. ARHGAP21 knockdown impairs erythroid commitment of human CMP and MEP. (A) Real-time PCR analysis of ARHGAP21 mRNA expression of FACS-sorted CMP, MEP, MkP and ErP (from 3 different donors) show upregulation of the transcript in MkP when compared to MEP (P = 0.04) and ErP (P = 0.04). (B–C) Knockdown of ARHGAP21 in CMP and MEP cells showing decreased (B) mRNA and (C) protein expression 24 h after transduction. (D) Improved dual CFU-Mk/E assay to determine Mk/E potential of transduced MEPs. Cells are plated in a collagen-based colony assay with EPO, TPO, SCF, IL-3, and IL-6 to promote growth of megakaryocyte and erythroid colonies. After 12 days of culture, colonies are stained for CD41a (Mk specific, false colored as blue) and GlyA (E specific, false colored as red) and checked for transduction efficiency (GFP+, green). Representative images of different colony types are shown. Typical CFU-Mk/E stains for both GlyA and CD41, BFU-E stains for GlyA only and CFU-Mk stains for CD41a only. Both Dual Mk/E of MEPs (E) and methylcellulose of CMPs (F) assays show decreased BFU-E when ARHGAP21 is knocked down (P = 0.002 in Anova tests for both assays). Results are presented as average + SD from 3 independent experiments.
	Fig. 5. RhoC activity is increased in Arhgap21+/− mice. (A-B) Active Rho A, RhoC and CDC42 was determined by GST-pull down assay from WT and Arhgap21+/− BM cells lysates. (A) Representative Western blots of Rhotekin-GST pull down (respective upper panel), assessment of total RhoA and RhoC (respective intermediate panels) and Actin (lowest panel). (B) Representative Western blots of PAK-GST pull down (upper panel), assessment of total CDC42 (intermediate panels) and Actin (lowest panel). (C) Quantification of western blot band intensities from GST-pull down assays normalized to the total amount of the respective RhoGTPase showing significant (P = 0.04) increase in RhoC activity.

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