CSF1 is essential for the expansion of macrophage colonies. (A) Membrane-bound Csf1 but not secreted Csf1 is prevalently expressed in IMECs by real-time PCR analysis. Ctrl, IMECs in the absence of coculture (n = 6). (B) Csf1 receptor (Csf1r) transcripts are highly expressed in colony cells (n = 6). (C) Immunolocalization of Csf1r (green) on a representative colony from day 10 coculture. DAPI indicates staining for nucleus (blue). Scale bar, 50 μm. (D) GW2580, a CSF1R inhibitor, significantly impairs colony emergence and growth. Coculture was treated with GW2580 (2μM) for 7 days. Ctrl indicates coculture exposed to vehicle. Arrow indicates a colony. Scale bar, 200 μm. (E) Quantification of colony cell number at day 14 of coculture in the presence or absence of GW2580 treatment (n = 3). (F) Representative dot plots showing increased apoptotic activity on GW2580 treatment. Colony cells from day 14 coculture treated with GW2580 (2μM) or vehicle were stained for propidium iodide (PI) and annexin V. (G) Apoptotic analysis of colony cells on GW2580 or vehicle treatment (n = 3; *P < .05; ***P < .001; unpaired Student t test).