Silencing of Gab2 but not Gab3 reduces CSF-1-dependent proliferation in 32D.R. (a) Gab2 knockdown with pU6 shRNA vectors. WT Gab2 was overexpressed in clone 24. Fifty micrograms was loaded except for WT Gab2 (5 μg). Growth curves and D4 viabilities were determined. The results are shown for 3 independent experiments. P values were calculated using the one-way analysis of variance (ANOVA) test. (b) Gab2 knockdown with shGab2 lentiviruses. Each CSF-1 MTS response curve is the average of 2 independent experiments. (c) Gab3 knockdown (shGab3) or combined Gab2 and Gab3 knockdown (Double) in 32D.R. Levels were normalized to the averaged control clones. Each CSF-1 response curve is averaged from 3 independent experiments. (b and c) MTS curves normalized to 0 to 1 for determination of EC₅₀. Control, thick black lines; shGab3, thin black lines; combined knockdown (“Double”), gray lines.

BM from Gab2^−/− mice has fewer macrophages and shows impaired CSF-1-dependent colony-forming activity. (a) Total BM cells were induced to differentiate along the MNP lineage. (b) Nucleated BM cells were stained with F4/80 antibody and analyzed by flow cytometry. The percentage of F4/80⁺ BM cells from individual mice is shown, with background staining from an isotype control subtracted from each measure- ment. High-side-scatter (SSC^hi) eosinophils, which also express F4/80 (58), were excluded as shown. (c) BM cells were stained with anti-CSF-1R. The percentage of CSF-1R⁺ cells in individual mice is plotted. (d) Total BM cells were plated in methylcellulose in the presence of CSF-1 and scored on D7. Photomicrographs of D10 colonies are shown. (e) D2 cells were seeded and scored on D7. D10 colonies are shown, and Wright-Giemsa staining was performed on cytospins prepared from the colonies. A single WT CFU-C was used per cytospin, whereas all visible Gab2^−/− CFU-C were pooled. In panels c and d, n = 3, with at least 5 mice/genotype.

Gab2 is required for optimal expansion of CSF-1-responsive colony-forming myeloid progenitors (LK31C). (a) Freshly isolated BM cells were lineage depleted and sorted for LIN⁻ c-Kit⁺ CD31^high Ly6C⁻ (LK31C) cells. Cytospins show blast-like morphology. (b) CFU-C in the presence of CSF-1 ± SCF. Each experiment used 3 mice per genotype. Cytospins from individual colonies were stained and analyzed by FACS. (c) Freshly isolated BM cells were stained with lineage antibodies and antibodies to Ly6C, c-Kit and CD31. The sequence indicated by arrows shows the hierarchical gating strategy. The LK31C fraction is shown as a percentage of total input cells. n, number of mice.

Impaired CSF-1-dependent proliferation and survival during MNP development in Gab2^−/− bone marrow. (a) Total BM cells were labeled with CFSE and either immediately processed (D0) or differentiated for 3 days. Gating was on the CD31⁺ subset. D0, both genotypes (thin black lines); D3, WT (black lines); D3, Gab2^−/− (gray shading). Corresponding geometric mean fluorescence intensities (MFI) are plotted. (b) D2 cells were labeled with CFSE and either immediately processed (D2) or differentiated. CFSE histograms are shown with representative plots of MFIs on D5 and D7 in the total and CD11b⁺ gates. The averaged MFIs in the total gate were normalized to WT. (c) Sorted LIN*⁻ CD31^high Ly6C⁻ cells were cultured in CSF-1 and analyzed for apoptosis and BrdU incorporation in the CD11b⁺ gate. (d) LK31C cells were immediately labeled after sorting with CFSE and cultured in CSF-1. Scatter plots, CFSE histograms, and the corresponding MFIs are shown. (e) A total of 5 × 10⁴ LK31C Flt3⁺ and Flt3⁻ cells were cultured in CSF-1, and cell counting was performed in duplicate (results from 1 of 2 experiments are shown).

Gab2 deficiency reduces Akt, Erk, and S6 phosphorylation in CSF-1-responsive BM progenitors. (a) LIN*⁻ CD31 and Ly6C subsets used in phospho-flow analysis. The LIN*⁻ CD31⁺ Ly6C⁺ subset was further segregated based on scatter (see text). Panels b to d show phospho histograms for the indicated subset at each time point (min) after CSF-1 addition. Inhibition by 20 μM LY and inhibition by 10 μM U0126 (U0) were used as controls for pAkt and pErk, respectively. Also included as controls are LIN*⁻ CD31⁻ Ly6C⁺ cells (WT or Gab2^−/−), which do not respond to CSF-1, and LIN*⁻ CD31⁻ Ly6C^high MOs (Gab2^−/−), which do respond. DMSO, dimethyl sulfoxide. The inset shows a plot of MFIs normalized to the WT signal at time 0. Data are representative of at least 3 independent experiments.

Gab2^−/− mice recruit fewer macrophages to the peritoneum after an inflammatory challenge, with an impaired BM CFU-C response. (a and b) Mice were injected with thioglycolate (TG). Peritoneal cells were counted and stained with CSF-1R and F4/80 antibodies to identify the macrophage population. At the time of sacrifice, total BM cells were also seeded for a CFU-C assay. (c) Mice were injected with LPS, and after 60 h, bone marrow was harvested for a CFU-C assay. (b and c) The differences in CFU-C between WT and −/− mice were significant at all time points. Data for individual mice are shown.

CSF-1 signals to Gab proteins in 32D.R cells (a) and BM-derived macrophages (b), as shown by immunoprecipitation (IP) and immunoblotting (IB) analyses. Previously identified tyrosyl phosphoproteins are indicated. An arrowhead points to the authentic Gab1 band that comigrated with tyrosine-phosphorylated Gab1. PY, phosphotyrosine; M, molecular mass markers (kDa).

Gab2 knockdown impacts CSF-1-mediated signaling in 32D.R cells. Separate immunoblots were performed for pAkt and total Akt (a), pErk and total Erk (b), and Gab2 (c). Shown are representative results for 1 of 2 independent experiments. Fold changes are relative to the phosphorylation of parental cells 5 min after CSF-1 addition.

Gab2 deletion is associated with decreased CD31^high Ly6C⁻ and CD31⁺ Ly6C⁺ cell numbers during in vitro MNP differentiation. (a) D2 cells were flow sorted into CD31 and Ly6C subsets. Cytospins stained with Wright-Giemsa revealed CD31^lo Ly6C⁻ lymphocytes (i), CD31^high Ly6C⁻ blasts (ii), CD31⁻ Ly6C⁺ granulocytes (iv), and CD31⁻ Ly6C^high MOs (v). The CD31⁺ Ly6C⁺ subset (iii) is a mixture of blasts, monoblasts, MOs, and Mφs. The CD31⁻ Ly6C^high subset was from Gab2^−/− cells. (b to d) Total BM cells were differentiated for 7 days and analyzed by FACS. (b) CD31⁺ Ly6C⁺ and CD31⁻ Ly6C^high gates are shown for D0 to D2, and the CD31⁻ Ly6C^high gate is shown for D5 to D7. Data were averaged from individual mice. (c) The data in panel b are shown as a percentage of the number of cells seeded 24 h prior to analysis (x), calculated by multiplying the percentage in the indicated subset by the total cell number present in the culture and dividing by x. The normalization accounted for the small differences in cell numbers seeded between experiments. (d) CD31⁺ CD11b⁺ gates are shown and data presented as in panel b. (e) D2 cells were lineage depleted to generate LIN*⁻ cells. Shown are representative CD31 and Ly6C plots of LIN*⁻ cells. The LIN*⁻ CD31⁻ Ly6C⁺ subset (vi) is Ly6G⁻ and CSF-1R⁻, indicating it is nonmonocytic and nongranulocytic. (e) Subsets from panel d were further gated on scatter. CSF-1R histograms are shown for the R0 gate in LIN*⁻ CD31^high Ly6C⁻ and the R2 gate in LIN*⁻ CD31⁺ Ly6C⁺: FMO, thin black lines; WT, black lines; Gab2^−/−, gray lines; and R1, thin gray lines. CSF-1R expression in the LIN*⁻ CD31 and Ly6C subsets is plotted as a percentage of all cells. (g) LIN*⁻ CD31^high Ly6C⁻ cells were sorted. Corresponding cytospins and CFU-C numbers are shown. Each experiment pooled cells from at least 3 mice of each genotype.

LK31C Flt3⁺ progenitors are enriched for CSF-1-responsive progenitors. (a) Immunophenotyping of LK31C Flt3⁺ and Flt3⁻ subsets. Shown are results representative of at least 3 independent experiments. (b) Sorted cells were cultured in CSF-1, and CSF-1R expression was determined by FACS. (c) Cells were plated in methylcellulose containing SCF, IL-3, IL-6, and erythropoietin (Epo). Representative colonies and their corresponding cytospins are represented as follows: CFU-M consists of exclusively Mφs, CFU-GM contains Mφs and granulocytes, CFU-mix contains Mφs, erythroid cells, megakaryocytes, and cells of the granulocytic lineage, and BFU-E contains erythroid cells. (d) CFU-C assay. Panels c and d show the results from 4 (WT) or 2 (Gab2^−/−) independent experiments; each experiment pooled cells from 5 mice/genotype.

Reexpression of WT Gab2 in Gab2^−/− BM progenitors completely rescues CFU-C formation, while Gab2 mutants deficient in PI3K or SHP2 recruitment are partially effective. (a) Murine stem cell virus (MSCV; empty vector control)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) retroviral constructs. LTR, long terminal repeat. (b) CFU-C assay of sorted GFP⁺ cells. CFU-C frequency is relative to total colony number from each experiment. (c) Photomicrographs of CFU-C. (d) CFU-C size and Wright-Giemsa stains of cytospins from CFU-C transduced with the indicated viruses. (b to d) Data were from 3 independent experiments. (e) CFU-C assay of WT and Gab2^−/− BM cells transduced with empty vector or WT Gab2, respectively, with or without LY294002 (LY). (f) CFU-C assay of sorted GFP⁺ cells. (g) Sorted cells were differentiated in liquid culture for 7 days, and Gab2 and Akt levels in BMMs were determined.

Gab2 deficiency increases CSF-1-induced JNK phosphorylation and reduces CSF-1-dependent proliferation in late-stage MNPs. (a) D5 BMMs were plated in CSF-1, and all cells were counted. The differences between WT and −/− cells are all significant. (b) D7 BMMs were stained with Hoechst 33258 and pyronin Y. Three independent experiments were performed. (c, left) D5 cells were seeded in 96-well plates and assayed after 48 h for MTS reduction. Four experiments were performed, each using cells from at least 2 mice/genotype. (Right) D5 cells were transduced with empty (MSCV) or WT Gab2 retrovirus. GFP⁺ cells were sorted and plated. Three transductions were performed. (d) Immunoblot analysis of BMMs stimulated with CSF-1. *, nonspecific band; **, probably cross-reacting Gab1 band. (e) Gab levels in D7 BMMs. M, molecular mass markers (kDa). (f) Mitogen-activated protein kinase (MAPK) phosphorylation in BMMs. The position of pJNK corresponds to p46 JNK1. The pJNK doublet has been noted previously (60). An arrowhead indicates the position of p54 JNK2. Shown are representative data from at least 3 independent experiments. (g) BMMs were deprived of CSF-1 (starved) or cultured in CSF-1 for 21 h. (h, top) BMMs were blotted with antibodies that specifically recognized JNK1, JNK1 and JNK2, or JNK2. (Bottom) BMMs were stimulated with LPS, and pJNK immunoblotting was performed. (i) MTS analysis of BMMs: L-JNKI-1, 0, 0.5, 1, or 2 μM; U0126, 0, 1, 5, or 10 μM. (j, top) Phospho-MKK7 immunoblot of BMMs. (Bottom) BMMs were pretreated with 0.5 or 2 μg/ml of actinomycin D for 10 or 15 min before CSF-1 addition. (k) Knockdown of JNK1 in BMMs. (Top) JNK1 levels in cells transduced with empty vector (v), shRNA-JNK viruses (sh1 to -4), with Stat5 levels as loading control. Quantification was based on 2 experiments (mean ± SD). (Bottom) MTS analysis of transduced BMMs. Shown are the results from 3 (i and ii), 4 (iii), or 5 (iv) separate transductions. Each experiment was performed in triplicate and with values expressed relative to the maximum value in WT cells transduced with empty virus.