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1.
Figure 4

Figure 4. Loss of microglia in CNS of DAP12-deficient mice. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

Immunohistochemical staining with the Iba-1 microglial marker in representative sections from the basal ganglia brain region (a,c) and spinal cord (b,d) of 10-month-old wild-type (a,b) and DAP12-deficient (c,d) mice. Arrows indicate some representative microglial cells. All panels are original 20× magnification and insets are original 60× magnification. Representative sections from one experiment with four mice per group are shown.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
2.
Figure 6

Figure 6. DAP12-deficiency augments MCSF-induced MAPK activation. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) BMDM were starved of MCSF for 4 h and then restimulated with MCSF (50 ng/ml). After the indicated times total cell lysates were prepared and subjected to immunoblot analysis using antibodies to the indicated phosphorylated proteins. To normalize the data the membranes were stripped and reprobed for the corresponding total proteins or actin. (b) BMDM were cultured in MCSF. At indicated times, cells were lysed and MKP-1 mRNA transcripts were measured by real-time PCR (mean and s.d.). *, P < 0.01 (Student’s t-test). Data are representative of three to five experiments.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
3.
Figure 5

Figure 5. Defect in BM repopulation by DAP12-deficient cells. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) BM from wild-type or DAP12-deficient mice was mixed 1:1 with BM from DAP12-sufficient GFP+ mice as shown in the histograms and adoptively transferred into irradiated wild-type hosts. (b) The GFP:GFP+ ratio in the BM of chimeras was determined after 12, 19 or 36 weeks of chimerism (mean and s.d.; n = 2-3 for 12 and 19 weeks, n = 6-7 for 36 weeks). (c) Identification of peripheral cell populations in mixed BM chimeras. Circulating PMN and monocytes (M) were identified by CD115Gr1hi and CD115+Gr1+ surface phenotypes, respectively. Peritoneal B cells (PB) were identified by CD19+ cells and peritoneal macrophages (PMac) by F4/80+ phenotypes. (d) GFP:GFP+ ratio in leukocyte populations from WT:GFP and DAP12-KO:GFP chimeras after 36 weeks of chimerism. Data are mean and s.d. of 4-7 individual mice; *, P < 0.05; #, P = 0.06 (Student’s t-test).

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
4.
Figure 1

Figure 1. Reduced yield of DAP12-deficient BMDM cultures. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) BMDM numbers were measured at indicated time points of culture of wild-type and DAP12-deficient BM cells with MCSF (mean and s.d.). *, P < 0.01 (Student’s t-test). (b) Propidium iodide (PI) analysis of the cell cycle and apoptosis of BMDM cultures at day 5, 7 and 10. Apoptotic cells (A, defined by cells with <2N DNA) and cells in the G1, S and G2 phases of the cell cycle were gated, and the percentage of total cells ± s.d. in each gate is indicated. *, P < 0.05 compared to wild-type at the same day of culture (Student’s t-test). Data are representative of three separate experiments each including two wild-type and two DAP12-deficient cultures.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
5.
Figure 2

Figure 2. Impaired proliferation of DAP12-deficient BMDM at day 5 of culture. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) Cell cycle status of wild-type and DAP12-deficient BMDM was measured by PI analysis 24 h after stimulation with 30% or 15% of L929-cell conditioned media (LCM) as a source of MCSF. The percentage of total cells ± s.d. in each gate is marked besides each histogram. *, P < 0.05 (Student’s t-test) (b) BMDM were transduced with retrovirus encoding DAP12-YFP at day 3 of culture. YFP+ and YFP cells were sorted 2 days later, re-plated and cultured for 24 h in medium containing 30% LCM. Cell cycle status was analyzed by PI staining as in a. (c) Proliferation of wild-type and DAP12-deficient BMDM was measured 24 h after incubation with BrdU in the presence of 30% LCM. (d) Expression of indicated mRNA transcripts in wild-type and DAP12-deficient BMDM were measured by real-time PCR at the indicated time of culture (mean and s.d.). *, P < 0.05, **, P < 0.01. (Student’s t-test) (e,f) wild-type BMDM were transduced with retrovirus encoding dominant negative DAP12-YFP (DAP12-DN) for 24 h. YFP+ and YFP cells were sorted, re-plated and cultured in medium containing 30% LCM. PI analysis of the cell cycle (e) and proliferation by BrdU incorporation (f) were assessed after 24 h. Data is representative of three (a,c,d) or two (b,e,f) experiments.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
6.
Figure 8

Figure 8. DAP12-mediated tyrosine phosphorylation of β-catenin involves Pyk2. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a,b) wild-type and DAP12-deficient BMDM or wild-type BMDM transduced with retrovirus encoding dominant negative DAP12 (DAP12-DN) were stimulated with MCSF (50 ng/ml). After the indicated times total cell lysates were subjected to immunoblotting analysis using antibodies to phosphorylated Pyk2. To normalize the data the membranes were reprobed for actin or total Pyk2. (c) wild-type BMDM were transduced with retroviruses encoding DAP12-DN or dominant negative Pyk2 (CRNK). Transduced cells were treated or not with MCSF 50 ng/ml for 5 min, total cell lysates were IP with antibodies to β-catenin and immunoblotted with antibodies to phosphotyrosine and β-catenin. Total cell lysates prior to IP were immunoblotted with anti-actin to control for protein amounts. (d) BMDM were stimulated with MCSF as in panel a. After 5 min, whole cell lysates were prepared and IP with anti-β-catenin, followed by immunoblotting analysis with Pyk2 or β-catenin-specific antibodies. Total cell lysates prior to IP were immunoblotted with anti-actin to control for protein amounts. (e) wild-type BMDM were cultured for 8 h in medium containing MCSF (LCM 15%) in the presence or not of the Pyk2 inhibitors PF431396 or AG17 (both at 5 μM). At the end of the incubation, BrdU incorporation (top) or cell cycle analysis (PI; bottom) was assessed. (f,g) Wild-type BMDM transduced with retrovirus encoding CRNK were cultured in medium containing MCSF for 24 h. PI analysis of the cell cycle (f) and BrdU incorporation (g) were assessed after 24 h. Data in e,f are mean ± s.d, *, P < 0.05 (Student’s t-test). Data are representative of three (a) or two (b-g) experiments.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
7.
Figure 7

Figure 7. MCSF activates the β-catenin signaling pathway in a DAP12-dependent manner. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) BMDM were stimulated with MCSF (50 ng/ml) for the indicated times and total cell lysates were analyzed by immunoblotting with anti-β-catenin and anti-actin. (b) Cell lysates were obtained as described in a and analyzed by immunoblotting with anti-phospho-GSK-3β Ser9 and anti-actin. (c) BMDM were treated with MCSF and cell lysates were subjected to immunoprecipitation (IP) with β-catenin-specific antibodies, followed by immunoblotting with antibodies to phosphotyrosine and β-catenin. Total cell lysates prior to IP were immunoblotted with anti-actin to control for protein amounts. (d) Cells were treated with MCSF and at indicated time points were lysed and divided into nuclear and cytoplasmic fractions. Graphs show densitometric analysis of β-catenin signals corrected for the quantities of housekeeping proteins (GAPDH for cytoplasmic fractions, lamin-B for nuclear fractions). (e) Cells were transduced with a retrovirus encoding a LEF/TCF-luciferase reporter and were stimulated with MCSF for the indicated time periods. (f) wild-type BMDM were transduced with retrovirus encoding dominant negative DAP12-YFP (DAP12-DN) and were stimulated with MCSF for the indicated time points. β-catenin nuclear accumulation was measured as in d. (g) BMDM were cultured in medium containing LCM 30% and BrdU in the presence of varying concentrations of the specific GSK-3β inhibitor SB216763 for 8 h, and the percentage of BrdU+ cells was assessed by FACS (mean and s.d.). *, P < 0.05 (Student’s t-test). Results are representative of three (a-c) or two (d-g) experiments.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.
8.
Figure 3

Figure 3. Impaired survival of DAP12-deficient BMDM. From: Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and ?-catenin.

(a) Representative histograms showing the percentage of apoptotic wild-type and DAP12-deficient BMDM after 24 h of MCSF deprivation. Numbers in histograms indicate percentage of hypodiploid (<2N DNA) cells, as measured by PI assay. (b) Cumulative percentages of apoptotic BMDM before and after MCSF deprivation (mean and s.d.). *, P < 0.01 (Student’s t-test) (c) BMDM were transduced with retrovirus encoding DAP12-YFP at day 3 of culture as described in Fig. 2. YFP+ and YFP cells were cultured for additional 24 h without MCSF. The percentage of total cells ± s.d. in each gate is marked below each histogram. *, P < 0.05 (Student’s t-test). (d) Pure cultures of myeloid precursors were generated as described in ref 27, then seeded at 105 cells/ml into 24 well plates, and cultured with MCSF for 48 h. Nonadherent cells were removed and the number of adherent macrophages enumerated (bar graph, day 0). Macrophages were cultured for 4 additional days in the indicated amount of LCM as a source of MCSF and the number of cells remaining was enumerated (mean and s.d.). (e) BMDM were cultured in the presence or absence of MCSF for 16 h; cell lysates were analyzed for the presence of cleaved (active) caspase-3 by immunoblotting. (f) GFP+ (wild-type) and GFP wild-type (WT), DAP12-KO, DAP10-KO or FcRγ-KO BM cells were mixed in a 1:1 ratio and co-cultured for 5 days in the presence of MCSF, followed by 2 days without MCSF. On day 7 the GFP+:GFP ratio was determined. Data are representative of five (a,b), two (c) or three (d-f) independent experiments.

Karel Otero, et al. Nat Immunol. 2009 July;10(7):734-743.

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