To901317

Primary mouse brain cells were obtained from the brains of C57BL/6J mice at postnatal day 3 using the Miltenyi Biotec Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-093-231) according to the manufacturer’s instructions. Cells were plated on 12 mm coverslips coated with poly-L-lysine (PLL) (Sigma Aldrich, P1274) at 70,000 cells per well for immunostaining and on 6-well plates at 3 × 10⁵ cells per well for western blot and cytokine expression analysis. The culture media consisted of DMEM/F-12 supplemented with 1% FBS, 1% penicillin‒streptomycin, N2, and 10 ng/mL PDGF-AA. Cultures were grown for 5 days before treatment with 10 µL of Nef or Ctrl EVs for 48 h. For drug treatment, cells were cultured for 7 days prior to receiving once-daily treatment with 1 µM of AMS-55 (generously provided by Dr. Amol Kulkarni) or TO-901317 (Sigma‒Aldrich, 575310) for 3 consecutive days. Subsequently, the cells were treated with EVs for 48 h. Post-treatment, the media were collected for cytokine analysis, while cells underwent analysis via western blotting or immunohistochemistry, as described below.

Peripheral blood mononuclear cells were isolated from a buffycoat (Sanquin blood supply, Amsterdam, the Netherlands) through density centrifugation using Lymphoprep™ (Axis‐Shield, Dundee, Scotland). Monocytes were then purified using human CD14 magnetic beads and MACS^® cell separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were plated in 24‐well tissue culture plates at a density of 1×10⁶ cells/mL (500 μL per well) and differentiated to macrophages for 6 days in Iscove's Modified Dulbecco's Medium (Sigma‐Aldrich) supplemented with 2 mmol/L l‐glutamine, penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% fetal calf serum (All Gibco, Waltham, MA) in the presence of 50 ng/mL macrophage colony stimulating factor (MCSF) (Miltenyi Biotec, Bergisch Gladbach, Germany). On day 3, the medium was removed and substituted by fresh Iscove's Modified Dulbecco's Medium with 10% fetal calf serum and 50 ng/mL MCSF. On day 6, all medium was removed and replaced by Iscove's Modified Dulbecco's Medium with 10% fetal calf serum without MCSF and cells were activated for 18 hours with vehicle (DMSO), LPS (10 ng/mL, Sigma‐Aldrich), a liver X receptor (LXR) agonist (TO‐901317, 10 μmol/L, Sigma‐Aldrich), and LPS+LXR agonist. Total RNA was extracted from the cell lysate.

Lipid droplet (LD) quantification was performed using FACS. Cells were collected and stained with 3.7 µM BODIPY for 30 min at room temperature (RT), protected from light. Single-cell data were acquired using Attune flow cytometer (Thermo Fisher Scientific) and analyzed using FCS Express 7 (De Novo Software). Gates were set up based on fluorescence minus one (FMO) controls. Gating strategy is provided in Supplementary Fig. 11. LXR agonist TO901317 was purchased from (Sigma-Aldrich, T2320), dissolved in DMSO to 0.01 M and used at 10 µM final concentration. LXR antagonist GSK2033 was purchased from (Sigma-Aldrich, SML1617), dissolved in DMSO to 5 mM and used at 2 µM final concentration.

The non-selective LXR agonist TO901317 was obtained from Sigma-Aldrich (T2320; China). MG132 was purchased from Calbiochem (474790, WI, USA). The FPR pan-antagonist N-tert-butoxycarbonyl-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe (Boc2) was obtained from MP Biomedicals (02152760; Solon, OH, USA). Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay kit was provided by Roche (11684817910, Basel, Switzerland).
The primary antibodies used in this study are shown in Table 1.

Insulin, TO901317, AA, and TSA were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-ACC, acetyl-histone H3, acetyl-histone H4, histone H2A, and HDAC5 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-SREBP1, α-Tubulin, HDAC1, HDAC2, HDAC7, and HDAC10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The immortalized murine BV-2 microglial cell line was purchased from the American Type Culture Collection (ATCC) and was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, United States) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, United States). Cells were plated in six-well flat-bottom plates at a density of 5 × 105/ml and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were passaged every 48 h with a 1:4 split ratio and used at passages 3–10. After a 24 h incubation, miR-155 mimic, miR-155 inhibitor or their respective controls (Gene Pharma Co., Ltd.) were transfected at a final concentration of 100 nM/ml in Opi-MEM (Life Technologies GmbH, Darmstadt, Germany) using Lipofectamine^® 2000 (Invitrogen, Life Technologies). Then, cells were treated with or without TO901317 for 1 h (10 nM, Sigma). Subsequently, cells were subjected to heat stress for 2 h in a prewarmed incubator at 42°C, followed by a recovery period at the normal growth temperature of 37°C. We collected the cell culture supernatant for enzyme linked immunosorbent assays (ELISAs) and extracted total RNA and protein for real-time PCR and Western blot analyses.