β-catenin nuclear accumulation was measured as in d. ( 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. ( e) Cells were transduced with a retrovirus encoding a LEF/TCF-luciferase reporter and were stimulated with MCSF for the indicated time periods. Graphs show densitometric analysis of β-catenin signals corrected for the quantities of housekeeping proteins (GAPDH for cytoplasmic fractions, lamin-B for nuclear fractions). ( d) Cells were treated with MCSF and at indicated time points were lysed and divided into nuclear and cytoplasmic fractions. Total cell lysates prior to IP were immunoblotted with anti-actin to control for protein amounts. ( 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. ( b) Cell lysates were obtained as described in a and analyzed by immunoblotting with anti-phospho-GSK-3β Ser9 and anti-actin. ( 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. Data are representative of five ( a, b), two ( c) or three ( d- f) independent experiments. On day 7 the GFP +:GFP − ratio was determined. ( 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. ( 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. 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.). Nonadherent cells were removed and the number of adherent macrophages enumerated (bar graph, day 0). ( d) Pure cultures of myeloid precursors were generated as described in ref 27, then seeded at 10 5 cells/ml into 24 well plates, and cultured with MCSF for 48 h.
![colony survival show colony survival show](https://cdn.pcgame.com/gen_screenshots/pcg/45910/screenshots/large/4-1920x1080.jpg)
in each gate is marked below each histogram. YFP + and YFP − cells were cultured for additional 24 h without MCSF. *, 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. ( b) Cumulative percentages of apoptotic BMDM before and after MCSF deprivation (mean and s.d.). Numbers in histograms indicate percentage of hypodiploid (<2N DNA) cells, as measured by PI assay. ( a) Representative histograms showing the percentage of apoptotic wild-type and DAP12-deficient BMDM after 24 h of MCSF deprivation.
![colony survival show colony survival show](https://occ-0-2794-2219.1.nflxso.net/dnm/api/v6/LmEnxtiAuzezXBjYXPuDgfZ4zZQ/AAAABQCC_eUyuI7HvXt4hTnrEqTGwTdPJbdVzjEgMqBcplKAZygbq5niHlVyv2qrbcxyxuMnui2ZFZoQruy3VxLnkHLX2MOGtrqyDT7I.png)
![colony survival show colony survival show](https://i.ytimg.com/vi/-Y1L4laiY5w/maxresdefault.jpg)
Data is representative of three ( a, c, d) or two ( b, e, f) experiments. PI analysis of the cell cycle ( e) and proliferation by BrdU incorporation ( f) were assessed after 24 h. YFP + and YFP − cells were sorted, re-plated and cultured in medium containing 30% LCM. (Student’s t-test) ( e, f) wild-type BMDM were transduced with retrovirus encoding dominant negative DAP12-YFP (DAP12-DN) for 24 h. ( 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.).
![colony survival show colony survival show](https://images2.minutemediacdn.com/image/upload/c_fill,g_auto,h_1248,w_2220/f_auto,q_auto,w_1100/v1555930819/shape/mentalfloss/the_colony_6.jpg)
( c) Proliferation of wild-type and DAP12-deficient BMDM was measured 24 h after incubation with BrdU in the presence of 30% LCM. Cell cycle status was analyzed by PI staining as in a. YFP + and YFP − cells were sorted 2 days later, re-plated and cultured for 24 h in medium containing 30% LCM. *, P < 0.05 (Student’s t-test) ( b) BMDM were transduced with retrovirus encoding DAP12-YFP at day 3 of culture. in each gate is marked besides each histogram. ( 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.