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. 2012 Oct 26;103(12):2165–2172. doi: 10.1111/cas.12015

Figure 5.

Figure 5

Involvement of macrophage colony‐stimulating factor receptor (M‐CSFR) in direct cell–cell interaction. (a) Membrane‐type M‐CSF (mM‐CSF) was expressed on the surface membranes of T98G cells. (b) T98G cells were cocultured with macrophages in the presence of anti‐M‐CSF antibody for 2 days, and interleukin (IL)‐10 in the supernatant was measured by ELISA. Non‐immunized rabbit IgG was used as the control. (c) Downregulation of M‐CSFR by siRNA was confirmed by real‐time PCR. (d) T98G cells were cocultured with macrophages having silenced M‐CSFR for 2 days, and IL‐10 in the supernatant was measured. (e) Macrophages and T98G cells were mixed and cultured with the M‐CSFR inhibitor GW2580 for 2 days, and IL‐10 in the supernatant was measured. (f) M‐CSFR of macrophages was silenced by siRNA, and coculture proceeded for 2 days. Signal transducer and activator of transcription‐3 (Stat3) activation in macrophages was evaluated by double immunostaining. (g) Macrophages were stimulated with M‐CSF (100 ng/mL) for 1 or 2 h, and Stat3 activation was evaluated. (h) M‐CSFR of macrophages was silenced by siRNA, and coculture proceeded. BrdU incorporation and Stat3 activation in T98G cells was examined by double immunostaining (n = 3 or 4 for each group).