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. Author manuscript; available in PMC: 2019 Apr 5.
Published in final edited form as: Nat Biomed Eng. 2018 Jul 2;2(8):589–599. doi: 10.1038/s41551-018-0254-6

Figure 2. AK750 inhibits CSF-1R and downstream signaling pathways in a sustained manner, and efficiently repolarize M2 macrophages to M1 phenotype.

Figure 2

(a) STRING map showing different direct or indirect protein interactions associated with CSF-1R signaling pathway in macrophages. The STRING map was generated by using STRING database version 10.0; (b) Schematic representation of CSF-1R pathways inhibition assay. RAW264.7 macrophage cells were pre-treated with either BLZ945, PLX3397 or AK750 for 4h, and then washed with cold PBS to remove the drugs that are not internalized. After 7h or 48h of recovery in fresh medium, the cells were stimulated with either MCSF for 2h. The cells were then washed and analyzed for activation of signaling pathway as shown in (c) Western blot for phosphor-CSF-1R, total CSF-1R and downstream signaling pathways. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. 13; (d) Schematic representation of macrophage repolarization assay. RAW264.7 macrophages were stimulated with IL4 for 24h, and then treated with either BLZ945, PLX3397 or AK750 for 12h before replacing with fresh medium. The cell lysates were collected at different timepoints for western blotting and FACS; (e–f) Quantification of flow cytometry data demonstrating expression of M2 markers (CD11b+CD206+), or M1 markers (CD11b+, MHC-II, CD86+ CD80+). The data shown are at (e) 12h, and (f) 72h time points. Statistical analysis was performed with One-way ANOVA with Newman-Keuls post Test. Data shows mean ± SEM (n = 3); ***p < 0.001; (g) Flow cytometry demonstrating expression of CD11b+CD206+, CD11b+MHC-II+, CD11b+CD86+ and CD11b+CD80+ on the macrophages at 72h time point following different treatments.