CD47 surface expression levels by various cell types and their corresponding ability to induce STAT3 phosphorylation in monocytes.

<p>(A) Different cell types were screened for surface CD47 expression using flow cytometric analysis. Grey histogram: isotype control. Black line: anti-CD47 staining. One representative experiment is shown. Comparable results were obtained in three independent experiments. (B) The average of three separate experiments is graphed as median fluorescence intensity (MFI) of CD47 staining. MFI was calculated by reducing the background isotype staining from the MFI value of anti-CD47 staining for each cell type. **- P≤0.001. (C) The various cell lines were co-cultured with monocytes for 2 h. Control cell extracts were obtained by incubating the cells and the monocytes separately and then mixing just before lysis. Cell extracts were subjected to SDS-PAGE and immunoblotting of anti-phosphorylated STAT3 (upper panels). Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Comparable results were obtained in at least two separate experiments for each cell type. Note: STAT3 phosphorylation in control lanes represents steady-state level of active STAT3 in the various cell lines. (D) Monocytes were either left untreated or treated with JSI-124 (10 µM) for 1 hour at 37°C, and then washed. Treated and untreated cells were then co-cultured with MCF7 and STAT3 phophorylation was determined as in B. (E) Monocytes were co-cultured with MCF7 cells for various time points and then lysed and analyzed as above. (F) Monocytes were co-cultured with MCF7 cells for 2 h in the absence or presence of 20 µM Actinomycin D, STAT3 phophorylation was determined as in B.