Fig 2: In vitro analysis of CSF1‐induced autophosphorylation of CSF1R mutant proteins. Representative western blots of lysates from HeLa cells that were transfected with wide‐type (WT) or mutant CSF1R expressing constructs with or without colony‐stimulating factor 1 (CSF1) treatment. Immunoblotting analysis using anti‐CSF1R antibody revealed comparable expression between WT and mutant CSF1R proteins. To evaluate the CSF1‐induced autophosphorylation activity of each CSF1R mutant, protein lysates were compared between CSF1‐treated and untreated cells expressing either one of the CSF1R mutant proteins. The five CSF1R mutants (T79M, K586*, G589R, R777Q, and R782C CSF1R) and the positive control M875T CSF1R had lost their ability to phosphorylate the Tyr546, Tyr699, Tyr723, and Tyr923 residues within CSF1R upon CSF1 stimulation, whereas the other three CSF1R mutants (E478K, T507_H508InsP, and A914T CSF1R) had preserved function of the CSF1‐induced autophosphorylation. Experiments were repeated three times with similar results. The equivalent amount of protein loading is shown using Actin as a loading control.

Fig 3: SYHA1813 influences the cell viability and differentiation of BMDMs and suppresses angiogenesis. (A) SYHA1813 influenced the CSF1-mediated cell viability of BMDMs. PLX3397 was used as a positive control. IC50 values were shown as mean ± SD from three independent experiments. (B) The phosphorylation of CSF1R and AKT in BMDMs was effectively inhibited upon SYHA1813 or PLX3397 treatment. (C, D) The expression of CD206 (C) and ARG1 (D) in BMDMs was decreased upon SYHA1813 treatment. Cells were stimulated with CSF1 (100 ng/mL), IL4 (10 ng/mL), and IL13 (10 ng/mL) and then treated with or without compounds. Flow cytometry was performed to detect the expression of CD206 and ARG1. The data were statistically analyzed by t-test from the results of three independent experiments (∗P < 0.05, ∗∗P < 0.01). (E) SYHA1813 inhibited the VEGF-mediated proliferation of HUVEC. IC50 values were shown as mean ± SD from three independent experiments. (F) Western blot analysis of HUVEC treated with or without compounds. SYHA1813 effectively inhibited the VEGF-induced phosphorylation of VEGFR-2 and the activation of downstream molecule ERK. Axitinib was used as a positive control. (G) SYHA1813 inhibited the tube formation of HUVECs (scale bars: 0.2 mm). (H) SYHA1813 inhibited the angiogenesis in the CAM model. Cover glass saturated with or without compounds was placed on the lower side of each field. Arrows indicate the edge line of the cover glass (scale bars: 2 mm).

Fig 4: Transcriptome analysis of N18-Fms cells stimulated by CSF-1. (A) Volcano plots showing all genes differentially expressed with p < 0.05 when comparing 24 h (left plot) and 48 h (right plot) of CSF-1 treatment with untreated control (0 h) (N = 2 for each group). Genes upregulated and downregulated with a fold change of 2 (shown by the grey line) or more after treatments are shown by red and green dots, respectively. The Spp1 gene is shown by a blue dot. (B) Venn diagram showing the intersection between genes upregulated (upper diagram) and downregulated (lower diagram) by the 24 h and 48 h CSF-1 treatments when compared to the untreated control. (C) Genes upregulated in both 48 h and 24 h of CSF-1 treatment when compared to untreated control (as shown in (B)) were subjected to a gene ontology enrichment analysis using the GO Biological Process (left panel) and Reactome terms (right panel). The −log10 of the FDR is shown for each enriched term with a FDR < 0.005 for Biological Process terms and a FDR < 0.01 for Reactome terms (shown by the dotted line). These thresholds were chosen for clarity purpose and the complete list of enriched terms is shown in Supplementary Table S1. (D) Same as in (C) using the genes downregulated in both 48 h and 24 h of CSF-1 treatment when compared to untreated control. All enriched terms with a FDR < 0.05 are shown.

Fig 5: Tumor volume evolution of xenografted prostate C2H subclones. (A) Each male SCID mice were injected subcutaneous with H9-Fms (left flank) and control H9 (right flank) cells, both cell populations stably expressing luciferase gene. Every 3 or 4 days, luminescence was measured in radiance (photon/second/cm2/steradians). (B) Photographic demonstration of comparative tumor size of 2 mice at day 17 (left panels) and day 31 (right panels). (C) Each male SCID mice were injected subcutaneous with N18-Fms (left flank) and control N18 (right flank) cells. Tumor volume was measured using calipers. (D) Forty-three days after cell injection, tumors were removed from 4 mice and photographed. For all these experiments, each group contained eight mice. The values are expressed as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001.