Nature Immunology: doi: /ni.1744

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1 Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and β-catenin Karel Otero, Isaiah R Turnbull, Pietro Luigi Poliani *, William Vermi *, Elisa Cerutti, Taiki Aoshi $, Ilaria Tassi, Toshiyuki Takai #, Samuel L. Stanley &, Mark Miller, Andrey S. Shaw and Marco Colonna Supplementary Figure 1. Differentiation of DAP12-deficient and WT BMDM. (a) BMDM were generated by culturing bone marrow cells with MCSF for 5 days. Cells were stained with antibodies for CD11b, F4/80, MHC II (I-A b ) and CSF-1R (CD115) and analyzed by flow cytometry. WT: gray profiles; DAP12- KO: thick line; isotype control: thin line. (b) RNA was extracted from BMDM and expression of CD36, ApoE and c-fms mrna transcripts was measured by realtime PCR. 1

2 Supplementary Figure 2. Similar monocyte/macrophage populations in blood and spleen of WT and DAP12-deficient mice. (a) Circulating PMN and monocytes were identified in WT and DAP12-KO blood based on CD115 - /GR1 hi and CD115 + /GR1 int-low surface phenotype, respectively. (b) Spleen macrophage populations were identified in spleen sections based on expression of different macrophage markers (ER-TR9, F4/80, MARCO and MOMA-1) (red). Sections were counterstained with B220 to identify B cells (green). Blue, phalloidin. 2

3 Supplementary Figure 3. Syk involvement in MCSF-induced BMDM proliferation. (a) DAP12-dependent activation Syk by MCSF. WT and DAP12- KO BMDM were cultured in the absence of MCSF for 4 h and then restimulated with MCSF (50 ng/ml). After the indicated times total cell lysates were prepared and immunoprecipitated (IP) with anti-syk. Immunoblots were performed with anti-phosphotyrosine. To control for protein loading, the membranes were stripped and reprobed for total Syk. (b) Syk contributes to MCSF-induced BMDM proliferation and cell cycle progression. WT BMDM were cultured for 8 h in medium containing MCSF (LCM 15%) either in the absence or in the presence of the Syk inhibitor (5 M). At the end of the incubation, BrdU incorporation (top) and PI analysis of cell cycle (bottom) was assessed. *, P < 0.05 by two-tailed Student's t-test. 3

4 Supplementary Figure 4. Attachment of WT and DAP12-deficient BMDM to fibronectin under static and continuous flow conditions. (a) Firm adherence of WT or DAP12-KO BMDM. Cells were plated for different times on fibronectincoated surfaces in the presence or absence of MCSF (50 ng/ml), washed and stained with Crystal Violet. (b) WT and DAP12-KO BMDM were left unstimulated (ns) or stimulated for the indicated times with MCSF. Dynamic cell adhesion on fibronectin-coated surface was measured at the shear stress of 1 dyne/cm 2. Results are shown as mean of cells bound to the substrate per field ± s.d. from three independent experiments. 4

5 WT DAP12-KO n.s. + MCSF Supplementary Figure 5. DAP12 deficiency does not affect MCSF-induced actin reorganization. WT and DAP12-KO BMDM were grown onto glass coverslips. Cells were starved from MCSF for 12 h and then left unstimulated (n.s.) or stimulated with MCSF (50 ng/ml) for 10 min. Cells were then fixed, stained with Texas Red-labeled phalloidin to visualize actin filaments and analysed by confocal microscopy. Representative immunofluorescence and phase contrast images at 60 magnification are shown side-by-side. Arrows, membrane ruffles; arrowheads, filopodia. 5

6 a WT DAP12 n.s. + MCSF b WT DAP12 n.s. + MCSF Supplementary Figure 6. DAP12 deficiency does not affect MCSF-induced focal adhesion and CD11b clustering. WT and DAP12-KO BMDM were plated, stimulated and fixed as described in Supplementary Fig. 5. (a) BMDM were stained with anti-vinculin. Peripheral stitching positive for vinculin, arrows. (b) BMDM were stained with anti-cd11b. CD11b clustering in membrane ruffles, arrows. 6

7 Supplementary Figure 7. MCSF stimulates phosphorylation of Pyk2 and increases β-catenin independently of adhesion. WT BMDM were stimulated with MCSF in suspension or after 1 h of adhesion on culture plates. Total cell lysates were analyzed by immunoblotting with antibodies to β-catenin, p-pyk2 or actin. 7

8 WT (cells 10 5 ) DAP12-KO (cells 10 5 ) Blasts 4.4± ±1.4 Committeed Precursors 18.7± ±7.5 Monocytes 25.8± ±5.9 PMN/Bands 45.5± ±13.8 Lymphoid 49.8± ±10.6 Peritoneal Mφ 9.2± ±6.2 Peritoneal B Cells 17.0± ±6.6 Supplementary Table 1. DAP12-deficient mice exhibit no defect in myelopoiesis. Bone marrow and peritoneal cells were isolated from WT and DAP12-KO mice. Bone marrow was stained for CD31 and Ly6c; peritoneal cells were stained for F4/80 and CD19. Blasts were defined as CD31 hi Ly6 ; committed precursors as CD31 + Ly6c + ; lymphoid cells as CD31 + Ly6c ; monocytes as CD31 Ly6c hi ; PMN as CD31 Ly6c +. Peritoneal macrophages were defined as F4/80 + CD19 ; peritoneal B cells as F4/80 CD19 +. Data are the average number of cells enumerated in 4-5 individual mice ± s.e.m. 8

9 Supplementary Table 2. Primers used for real-time PCR. name Forward Reverse c-myb 5 -GAATAAAGGAGCTGGAGTTGCTC-3 5 -GTGCATCTAAGCCCGAGCTTTC-3 c-myc 5 -AATCCTGTACCTCGTCCGAT-3 5 -TCTTCTCCACAGACACCACA-3 Cyclin D1 5 -TGCTACCGCACAACGCA-3 5 -TCAATCTGTTCCTGGCAGGC-3 Cyclin D2 5 -CGTGTGATGCCCTGACTGAG-3 5 -GACTTGGATCCGGCGTTATG-3 MKP-1 5 -CTCCAAGGAGGATATGAAGCG-3 5 -CTCCAGCATCCTTGATGGAGTC-3 CD36 5 -TCCAGCCAATGCCTTTGC-3 5 -TGGAGATTACTTTTCAGTGCAGAA-3 ApoE 5 -TGTTTCGGAAGGAGCTGACT-3 5 -TGTGTGACTTGGGAGCTCTG-3 c-fms 5 -GCGATGTGTGAGCAATGGCA-3 5 -CGGATAATCGAACCTCGCCA-3 HPRT1 5 -GCAGTACAGCCCCAAAAT-3 5 -AACAAAGTCTGGCCTGTATCCAA-3 9

10 Supplementary Methods Adhesion assays For firm adherence, BMDM ( ) were seeded onto 96-well plates coated with 3 g/ml fibronectin (Sigma) overnight at 4 C. Cells were incubated for 2, 5 and 10 min with or without MCSF, then washed twice with DMEM (HyClone). Cells were fixed in methanol for 2 min, stained with 0.5% crystal violet (Sigma) in H 2 O for 5 min and rinsed 5 with distilled water. 100 µl of 1% SDS was added to each well to solubilise the dye. Absorbance was measured at 595 nm. For adhesion under shear stress, tissue culture plates (35 mm; Corning Life Sciences) were coated with fibronectin as described above. These plates were used with a parallel plate flow chamber (Glycotech) and PHD 2000 infusion pump (Harvard Apparatus). BMDM ( ) were resuspended in PBS-5% FCS, 1 mm MgCl 2 and 1mM CaCl 2 and were either left untreated or stimulated with MCSF. Cells were then flowed at a force of 1 dyne/cm 2. Attached cells were visualized using the 10 lens of a light microscope and camera (AmScope). The number of cells adhering to the plate was immediately determined in 15 different fields. Immunofluorescence BMDM were plated on glass coverslips (Fisher Scientific) and grown to 60-70% confluence in the presence of MCSF. For MCSF stimulation experiments, cells were starved from MCSF for 12 h. After incubation with MCSF (50 ng/ml) cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilised with 0.5% Triton X-100 for 30 min, and blocked with 2% FCS, 4% BSA in PBS for 1h. The following antibodies were used: monoclonal anti-cd11b-fitc (M1/70, BD Pharmingen), monoclonal anti-vinculin (VIN-11-5, Sigma) visualized with Cy3- conjugated secondary antibody (Jackson ImmunoResearch). F-actin was visualized using texas-red-labelled phalloidin (invitrogen). Specimens were mounted in PBS-15% Mowiol (Calbiochem)-50% glycerol. Confocal laser scanning microscopy was carried out with an Olympus 1 81 inverted microscope with 60 oil immersion objective and analyzed with Olympus Fluoview 1.7a software. 10