Pexidartinib

MyC-CaP (ATCC, Manassas, VA, USA) and RAW264.7 (RAW) (National Collection of Authenticated Cell Cultures at Shanghai, China) were cultured in DMEM media supplemented with 10% charcoal stripped fetal bovine serum (FBS), which mimics the low-androgen condition in castration-resistant prostate cancer, and 1× Penicillin–Streptomycin. PC-3 (National Collection of Authenticated Cell Cultures at Shanghai, China) and C4-2 (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 media supplemented with 10% FBS and 1× Penicillin–Streptomycin. All cells were cultured in 37 °C, 5% CO₂ humidified cell incubators. Pexidartinib was purchased from Selleck (Cat#S7818, Shanghai, China). The overexpression plasmid of murine CXCL12 was developed through Gibson assembly of the PCR product from the forward primer 5′-CGCCAGAACACAGGACCGGTTCTAGAATGGACGCCAAGGTCGTC-3′ and the reverse primer 5′-CATCGTCTTTGTAATCCATCTCGAGCTTGTTTAAAGCTTTCTCCAGGTAC-3′, and the double digested plasmid lentiCRISPRv2 hygro from Addgene (Cat#98291, Watertown, MA, USA) was developed with Gibson assembly mastermix (Cat#E2611S, NEB Biolabs, Ipswich, MA, USA). The ELISA kit used to examine the CXCL12 from RAW cells was purchased from R&D Systems (Cat#MCX120, Minneapolis, MN, USA), and ELISA was undertaken in accordance with the manufacturer’s protocol.

PLX3397 (Pexidartinib; Selleckchem, USA) was formulated in AIN‐76A standard chow (FBSH Biopharmaceutical Co., Ltd., Shanghai, China) at a concentration of 290 mg/kg. Normal AIN‐76A diet was served as the control.
Mice were fed with a PLX3397‐formulated diet (PLX3397) for 7, 10, 14 and 21 days to determine the optimal days for microglial elimination and repopulation in the nigrostriatal pathway (n = 3 per group). After 21 days, a subset of mice with the PLX3397 diet were switched to a control diet (CD) for 4 and 7 days (n = 3 per group) to repopulate microglia.
Mice were fed with a PLX3397 diet or a control diet for 21 days and followed by a diet according to the requirements of different experiments to clarify the function of microglia in PD (n = 4‐5 per group). The design of each experiment was shown as a diagram, respectively.

Human melanoma cell line M230 was provided by Pr A. Ribas and was cultured in RPMI 1640 (Invitrogen, Cergy Pontoise, France) containing 10% (v/v) fetal calf serum (FCS; Perbio, Bredières, France), L-glutamin (2 mM; Gibco, Cergy pontoise, France), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco). Human melanoma cell lines HBL and LND1 were provided by Pr G E Gahnem and were cultured in F10 (Invitrogen) containing 10% (v/v) fetal calf serum (FCS; Perbio), L-glutamin (2 mM; Gibco), antibiotics (100 U/mL penicillin and 1000 μg/mL streptomycin; Gibco). Imatinib, nilotinib, sorafenib, dasatinib, pexidartinib, and trametinib were from Selleck Chemicals, dissolved in DMSO and used at 1 µM final concentration for KIT inhibitors and 0.2 µM for trametinib. FGF2 was from PeproTech and used at 20 ng/mL final concentration.

The CSF1R inhibitor pexidartinib (Selleck Chemicals) was used to deplete macrophages. pexidartinib was initially suspended and stored at 200 mg/mL in DMSO, and further suspended in 5% DMSO, 45% polyethylene glycol 3000 (Sigma-Aldrich), 5% Tween-80 (Sigma-Aldrich), and 45% ddH₂O for a working concentration of 10 mg/mL. FVB/N mice bred in-house at 5 weeks of age were given a working solution of pexidartinib at 45 mg/kg once daily by oral gavage for 2 weeks. Mammary glands were then either fixed in 4% paraformaldehyde for histology, processed for flow cytometry or lysed for HA analysis by ELISA as previously described (Bohrer et al., 2014 (link)). HA concentrations were normalized to mammary gland weight.