Fig 4. SOD3_R213G expressing neutrophils exhibit altered G-CSF mediated signaling, promoting proliferation, apoptosis, and trafficking.

A and B. The expression of SOD3_R213G and G-CSFR in neutrophils. The expression of the indicated genes was assessed by RT-PCR (A) and immune blot (B) as described in Materials and Methods. C. Down regulation of SH-PTP1 signaling in neutrophils of 17 week old SOD3_R213G mice. G-CSF mediated Src tyrosine phosphatase, SH-PTP1 level (C, Top panel) was assessed by treating the neutrophils isolated from 17 week old SOD3_R213G or Wt mice with G-CSF (20 ng/ml) at the indicated time. The cells were lysed and subjected to SDS-PAGE, followed by immune blot with an antibody against SH-PTP1. D-F. Proliferation, apoptosis, and chemokine receptor CCR2 expression in neutrophils. The proliferation (D) and apoptosis (E) of neutrophils were measured by treatment with G-CSF (100 ng/ml) for 5 days as described in Materials and Methods. Chemokine receptor CCR2 expression (F) in neutrophils was assessed by qRT-PCR. G and H. Granulopoiesis and maturation of neutrophils were not affected by SOD3_R213G. To assess granulopoiesis (G), BM isolated neutrophils from SOD3_R213G or Wt mice were subjected to FACS analysis with the indicated antibodies and myeloid progenitor cells were assessed. GMP: granulocyte-monocyte progenitors, CMP: common myeloid precursors, MEP: megakaryocyte-erythrocyte precursors. For the maturation analysis (H), CD11b^lowGr1^high cells for immature and CD11b^highGr1^high cells for mature neutrophils were assessed by FACS analysis. RT-PCR, immunoblot, and FACS data are representative of at least three independent experiments. All qRT-PCR data were analyzed as described in Fig 1I–1M. Statistical analysis was performed by t- test (*p<0.001).