p85α subunit of class IA PI-3 kinase is crucial for macrophage growth and migration by Veerendra Munugalavadla, Jovencio Borneo, David A. Ingram, and Reuben Kapur Blood Volume 106(1):103-109 July 1, 2005 ©2005 by American Society of Hematology

Deficiency of p85α in BMMs results in reduced proliferation. Deficiency of p85α in BMMs results in reduced proliferation. Nonadherent bone marrow cells (1 × 106) from WT and p85α–/– mice were cultured in a 24-well tissue-culture plate in the presence of M-CSF (100 ng/mL) for 3 days. Cells were starved overnight using IMDM and stimulated with either M-CSF (100 ng/mL) or GM-CSF (50 ng/mL) for 24 hours. Proliferation was measured by thymidine incorporation assay. Bars represent the mean thymidine incorporation in BMMs in response to either M-CSF or GM-CSF stimulation (counts per minute [cpm] ± SD) of 2 experiments performed in duplicate. Similar results were observed in 3 independent experiments. *P<.05 for WT versus p85α–/–. Veerendra Munugalavadla et al. Blood 2005;106:103-109 ©2005 by American Society of Hematology

Deficiency of p85α in BMMs results in reduced adhesion and migration. Deficiency of p85α in BMMs results in reduced adhesion and migration. (A) Expression of Mac-1 on WT and p85α–/– BMMs. BMMs were stained with an anti–Mac-1 antibody or an isotype control antibody and subjected to flow cytometric analysis. Filled histograms indicate the level of Mac-1 staining on the surface of both WT and p85α–/– BMMs; open histograms, the level of staining using an isotype control antibody. (B) WT or p85α–/– BMMs (5 × 105) were subjected to an in vitro adhesion assay on fibronectin fragments CH271 (which contains a binding site for α5β1), H296 (which contains binding sites for α4β1), and CH-296 (which contains binding sites for both α4β1 and α5β1) and VCAM-1. Adhesion was assessed by measuring absorbance at indicated times. Shown is the optical density of the adherent cells at 600 nm. *P<.05 for WT versus p85α–/–.WTor p85α–/– BMMs (2.5 × 105) were subjected to an in vitro migration assay on fibronectin fragment CH271 (which contains a binding site for α5β1), H296 (which contains binding sites for α4β1), and CH-296 (which contains binding sites for both α4β1 and α5β1) as well as VCAM-1–coated transwell membranes. Cell migration was performed either in the absence (C) or presence (D) of M-CSF. Cell migration is expressed as the average number of cells migrated. Figure shows the average number of cells migrated ± SD. Ten fields were scored from one representative experiment. Similar results were observed in 3 other experiments. *P<.05 for WT versus p85α–/–. Veerendra Munugalavadla et al. Blood 2005;106:103-109 ©2005 by American Society of Hematology

Impaired migration of p85α–/– BMMs in a wound-healing assay. Impaired migration of p85α–/– BMMs in a wound-healing assay. (A) WT and p85α–/– BMMs were cultured for 8 days in 24-well plates in IMDM/20% FBS/100 ng/mL M-CSF. An artificial wound was created in the macrophage monolayer using a pipette tip. Photographs were taken immediately and again at indicated time periods after creating the wound. Data are from one representative experiment. Similar results were obtained in 2 independent experiments. Images were acquired through an Olympus TH3 microscope equipped with a 20×/0.4 objective lens (Olympus, Melville, NY), and were captured with a Nikon HRD100 camera (Nikon, Melville, NY). (B) Quantification of the wound-healing assay. The figure shows the mean number of cells migrated ± SD. Data are from one representative experiment. *P<.05 for WT versus p85α–/–. Veerendra Munugalavadla et al. Blood 2005;106:103-109 ©2005 by American Society of Hematology

Reduced phagocytosis in p85α–/– BMMs Reduced phagocytosis in p85α–/– BMMs. Phagocytosis assay was performed using WT and p85α–/– BMMs as described in “Materials and methods.” Phagocytosis assay was initiated by subjecting BMMs to IgG-sensitized sRBCs at a target-effector ratio of 100:1 for 15 ... Reduced phagocytosis in p85α–/– BMMs. Phagocytosis assay was performed using WT and p85α–/– BMMs as described in “Materials and methods.” Phagocytosis assay was initiated by subjecting BMMs to IgG-sensitized sRBCs at a target-effector ratio of 100:1 for 15 minutes at 37°C. Nonengulfed sRBCs were lysed by water shock treatment and cells were fixed and stained using Diff-Quik stain before measuring the phagocytic index. (A) Arrows indicate engulfed cells. Images were acquired through a Zeiss Axioskop 2 Plus microscope equipped with a Plan-Neofluar 20×/0.5 objective lens, and were captured with an Axiocam MRC-5 camera and Axiovision 4 software (all from Zeiss, San Marcos, CA). (B) Bars indicate the mean phagocytic index ± SD. Data are from 4 independent experiments. *P<.05 for WT versus p85α–/–. The ability of WT and p85α–/– BMMs to bind IgG-sRBCs was assessed as described in “Materials and methods.” (C) Shown is the percent of BMMs bound to IgG sRBCs ± SD. (D) Expression of FcγIII/II receptor on WT and p85α–/– BMMs was analyzed by using an anti-FcγIII/II receptor (CD16/32) antibody or an isotype control antibody and flow cytometric analysis. Solid histograms indicate the level of FcγIII/II receptor staining on the surface of both WT (97.94% positivity) and p85α–/– (98.33% positivity) BMMs. Open histograms show the level of staining using an isotype control antibody. Veerendra Munugalavadla et al. Blood 2005;106:103-109 ©2005 by American Society of Hematology

Reduced activation of Akt and Rac in p85α–/– BMMs Reduced activation of Akt and Rac in p85α–/– BMMs. Wild-type and p85α–/– BMMs were starved overnight and stimulated with M-CSF. Reduced activation of Akt and Rac in p85α–/– BMMs. Wild-type and p85α–/– BMMs were starved overnight and stimulated with M-CSF. Equal amounts of cell lysates were subjected to Akt and Erk MAP kinase activation (A) by Western blot analysis using an anti–phospho Akt or Erk1/2 antibody or to a p21-activated kinase (PAK)–binding pull-down assay (B), which measures active, GTP-bound Rac. Shown are representative blots. Three independent experiments showed similar results. The bars indicate the band intensity of activated Akt and Rac, expressed as arbitrary units. Veerendra Munugalavadla et al. Blood 2005;106:103-109 ©2005 by American Society of Hematology