Plx3397

First, 5* 10^6 CCLP-1 cells suspended in 100uL PBS were injected subcutaneously into the flank of female BALB/c athymic nude mice (4–6 weeks old). When tumors reached a volume of 0.2-0.3 cm³, mice were divided into 4 groups (5 mice per group): Blank, Zileuton (Selleck), PLX3397 (Selleck), Zileuton + PLX3397. Zileuton: Zileuton: 200 mg/kg/day by oral gavage daily for 28 days; PLX3397: 50 mg/kg/2 days by oral gavage every other day for 14 days. After 4 weeks of continuous treatment, the tumor size and number of mice in each group were detected, and the abundance of ALOX5 and macrophages were observed by multiplex immunofluorescence technique and flow cytometry. Tumor volume was calculated using the following formula: Volume = (length * width²)/2.

Primary mixed cortical and hippocampal neural cultures were isolated as described elsewhere.^{20 (link),21 (link)} Cortices/hippocampi were isolated from postnatal day 1 to 2 pups from TgP301S mice. Meninges were removed and cortices and hippocampi digested with trypsin (0.25% wt/vol) and DNAse (0.1% wt/vol, Thermo Fisher Scientific, 50-443-716). Neurons were dissociated by trituration and plated onto precoated poly-L-lysine (0.1 mg/mL, R&D Systems, Cultrex 3438-100-01) plastic ware at a density of 30,000/well in 96 wells in plating medium consisting of Neurobasal Plus (Gibco, A3582901), GlutaMAX (1 mM, Gibco, 35050061), horse serum (10%), penicillin-streptomycin (1%), and 1X B-27 Plus growth factor supplement (Gibco, A3582801). Three hours after plating, medium was exchanged for maintenance medium (plating medium devoid of horse serum). At DIV6, the cultures were topped up with maintenance medium (50% volume). When L-leucine methyl ester (LME)-based microglia depletion was conducted, at DIV6, 15 mM LME was added to the medium for 4 h, and a full medium change into maintenance medium was conducted subsequently (as described in ref. [22]). When PLX3397-based microglia depletion was conducted, 2 μM PLX3397 (SelleckChem, S7818) was added to the medium at DIV0 and supplemented repeatedly every 48/72 h until DIV14.

5xFAD mice (Tg6799; Jackson Laboratory, stock #006554) and JNPL3 mice (TauP301L‐JNPL3; Taconic, Stock#2508 homozygote) were crossed to produce ADLP mice as described (Kim et al., 2018 (link)). At 6 months old, ADLP mice were randomly allocated into saline or FA‐treated groups. Flufenamic acid (Supelco, St. Louis, MO) or saline (DMSO added to an equal volume with flufenamic acid compound) was administered intraperitoneally, once a day, for 8 weeks. For microglia‐depleted condition, PLX 3397 was purchased from Selleckchem (Houston, USA) and formulated in chow (AIN‐76A) at 300 mg/kg. Mice were treated with PLX or control diet for 12 weeks. Intraperitoneal injection of saline or FA was started after 4 weeks of chow treatment. 6–10 mice were used for each group, and they were killed after behavioral test. Brain samples of mice were randomly allocated for staining or Western blot. The number of mice used for each experiments is written in legends of each figure. Animals were treated and maintained in accordance with the Animal Care and Use Guidelines of Seoul National University, Seoul, Korea.

All mouse experiments were approved by the NICHD Animal Care and Use Committee Protocol #15-002 and #18-002. BALB/c-Npc1^+/− were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Heterozygous Npc1^+/nih mice (BALB/cNctr-Npc1^m1N/J strain) [103 (link)] were bred to obtain control (Npc1^+/+) and mutant (Npc1^−/−) littermates. Mice genotype were identified by PCR using the primers listed in Table S3. Water and mouse chow, control or with 290 mg·kg⁻¹ PLX3397 (Selleckchem, Houston, TX, USA), were available ad libitum. Genotyping PCR was performed using ear punch DNA. A humane survival endpoint was defined as hunched posture, reluctance to move, inability to remain upright when moving, and weight loss >30% of peak weight.

Rotenone (#R8875) and Evans blue (#E2129) were provided by Sigma-Aldrich, Inc. (St. Louis, MO, USA). The AG RNAex Pro Reagent (#AG21102), Pro Taq HS qPCR Kit, and SYBR Green Premix (#AG11720) were provided by Accurate Biotechnology (Hunan, China). The PLX3397 (#S7818), minocycline (#S4226), and SB-3CT (#S7430) were provided by Selleck (Shanghai, China). Antibodies against tyrosine hydroxylase (TH, #AB152) and ionized calcium binding adaptor molecule-1 (Iba-1, #019-19741) antibodies were provided by EMD Millipore (Temecula, CA, USA) and Wako Chemicals (Richmond, VA, USA), respectively. Antibodies against fibrinogen (#ab34269), zonula occludens-1 (ZO-1, #ab96587), claudin-5 (#ab131259), occludin (#ab167161), MMP-2 (#ab92536), MMP-9 (#ab38898), GAPDH (#ab181602), and the MMP assay kit (#ab112146) were purchased from Abcam (Cambridge, MA, USA). The ECL reagents (#20-500-120) were provided by Biological Industries (Cromwell, CT, USA).

As previously described,^{34 (link)} PLX3397 (Selleckchem, Houston, TX) was formulated in AIN-76 A standard chow at 40 mg/kg/day for 21 consecutive days prior to middle cerebral artery occlusion (MCAO) surgery and continued until the end of experiments. AIN-76 A standard chow alone served as the control. Bindarit (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3-yl]methoxy] propanoic acid), an inhibitor of monocyte chemotactic protein synthesis, was diluted in 0.5% carboxymethylcellulose aqueous solution, and administered at a dose of 50 mg/kg by oral gavage, twice daily. Bindarit treatment was initiated immediately before MCAO and continued until the end of experiments. Control animals received an equal volume of carboxymethylcellulose.

PLX3397 was purchased from SelleckChem (S7818, China) and formulated in AIN-76A standard chow by Shanghai biopikeChem at 290 mg/kg doses.