Csf1r

Cell pellets were resuspended in a cell lysis buffer (Cell Signaling Technology Inc.). After centrifugation, the protein content of the supernatants was determined using a Bradford Protein Assay Kit (Bio‑Rad Laboratories Srl.). Then, 60 mg of protein was prepared in a sample buffer containing 10% SDS, 1.0 M Tris-HCl pH 6.8, 8% glycerol, and 0.05% (w/v) bromophenol blue. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated overnight with the following primary antibodies: CSF1R (ab205921; Abcam); BAX (mouse anti‑human BAX antibody; 610,983; Becton Dickinson Holdings Pte. Ltd.); BCL‑2 (rabbit anti‑human BCL‑2 antibody; ab196495; Abcam); and β‑actin (mouse anti-β‑actin antibody; sc‑47,778; Santa Cruz Biotechnology Inc.). Further incubation was undertaken with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The signals were visualized by ECL (Thermo Fisher Scientific Inc.) under Gel Document Syngene (Syngene), and the bands were quantified by ImageJ (version 1.48v; National Institutes of Health, Bethesda, MD, USA). The densitometric values of all protein bands were normalized to that of β-actin and quantified using ImageJ (version 1.52a).

Slides from tumors were deparaffinized in xylene and dehydrated in graded ethanol solutions. The sections were blocked in goat serum blocking solution for 1 h at room temperature. The slides were incubated overnight at 4 °C with the following antibodies for multicolor immunofluorescence staining: F4/80 (Cell Signaling Technology, 30,325 T, 1:200), CD206 (Proteintech, 18,704–1-AP, 1:200), Arg1 (Abcam, ab96183, 1:100), iNOS (Abcam, ab178945, 1:200), CD8 (Bioss, bs0648R, 1:100), aSMA (Cell Signaling Technology, 19,245, 1:200); Ki67 (Cell Signaling Technology, 12,075, 1:100); CSF1R (Abcam, ab254357, 1:100) and Granzyme B (Cell Signaling Technology, 17,215, 1:200). Immunofluorescence staining was detected with fluorescence microscope.

The binding affinities of CPPC and ligand 1 for CSF1R were measured using a commercially available assay kit (Thermo Fisher Scientific, Z’-LYTE kinase assay kit-tyrosine 1 peptide) and CSF1R (Abcam, Cambridge, UK). CPPC or 1 was dissolved in DMSO and serially diluted and then added to the reaction buffer that contained 50 mM HEPES, 0.01% BRIJ-35, 10 mM MgCl₂, 1 mM EGTA, 1 mM ATP, and CSF1R (1 ng). The final concentration of DMSO in the incubation mixture was 1%. The mixtures were incubated at room temperature for 1 h, followed by development reaction incubations at room temperature for 1 h. The fluorescence intensity of the incubation mixtures was measured (excitation, 400 nm; emission, 445 nm (donor) and 520 nm (acceptor)) using a Mithras² LB 943 monochromator multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany), and the phosphorylation extent of the FRET-peptide was calculated. All experiments were performed in triplicate, and the IC₅₀ values represent the average of three experimental determinations.