Transfection of cell lines was performed by generating plasmid DNA/polyethylenimine complexes using methods modified from Volasertib previous studies (23). Stable cell lines were generated and characterized as described previously (2) or by transducing cells with amphotropic retrovirus, followed by selection in puromycin (5 ��g/ml) for 3 d. For cells expressing fluorescently tagged proteins, stable cell lines were generated by flow cytometric sorting of highly fluorescent cells 14 d after transfection or 4 d after viral transduction. To induce autophagy, cells were amino acid starved by washing 3 times with PBS, then cultured in either HBSS with 1 mM HEPES or with rapamycin (400 nM) in serum-free medium for 6 h. Mouse bone marrow-derived macrophages (BMMs) were cultured from whole bone marrow (BM) as described previously and used between d 7 and d 10 (1).

Ms were purified from kidney, using methods previously described (3, 19). The single-cell suspension of kidney cells was incubated 1 h in serum-fee medium on glass cover slips. Nonadherent cells were then removed by vigorous washing 5 times prior to fixation and immunolabeling. For phagosome scoring assays, cultured cells were imaged at ��400 in a masked fashion. Images were assessed in a masked fashion for number of labeled endosomes per cell. Phagocytosis assays were carried out in 12-well plates using CMFDA-labeled apoptotic thymocytes. After washing away noningested apoptotic cells, live cells were assessed by flow cytometry as described previously (2). Apoptotic thymocytes were generated by dexamethasone treatment as described previously (1).

For imaging studies, cells were cultured on glass cover slips with or without apoptotic thymocytes in a 10:1 ratio for 1 h or zymosan particles (100 ��g in 200 ��l) for 1 h or polystyrene beads (0.5 ��m) (Polysciences, Warrington, PA, USA). Cells were washed 5 times with ice-cold PBS cells and fixed as described above. To quantify degradation of apoptotic thymocytes in BMMs, subsequent to removal of noningested apoptotic cells by washing with PBS, BMMs were returned to 37��C incubator for 1, 2, 4, 8, or 24 h and then lysed. To quantify autophagy, LC3-II was identified by immunofluorescence (see below) or by the pattern of expression of GFP-LC3. Cells were quantified as autophagic if they had LC3-II vesicles.

Batimastat To assess lysosomal fusion with phagosomes, LysoTracker Red (Molecular Probes, Eugene, OR, USA) was applied to cells (30 min, 37��C, 200 nM). Amphotropic retroviruses were generated as follows: 293T17 cells in 100-mm dishes were cotransfected with pCLampho (Imgenex, San Diego, CA, USA) and pMSCV-IRES-GFP, pMSCV-Gpnmb-IRES-GFP, pMXs-puro, or pMXs-GFPLC3-puro in equimolar concentrations. After 16 h, virus was collected in 6 ml of M medium or other cell culture medium for 6 or 24 h. The virus was harvested and filtered through a 0.45-��m filter.