Penicillin 100

The H1975, H358, HCC827, A549 and H1650 LADC cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were cultured in RPMI-1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma) and 100 U/ml penicillin-100 Ag/ml streptomycin (Sigma). HepG2 hepatoma cells were cultured in MEM supplemented with 1 mM sodium pyruvate, 1% non-essential amino acids and 10% FBS). All cell lines were maintained at 37°C in a humidified incubator with 5% CO₂.

BV2, Neuro-2a (N2A) cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 100U penicillin/100 g streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Procell Life Science & Technology Co., Ltd. provided BV2 (Procell CL-0493). Shanghai Zhongqiaoxinzhou Biotech supplied N2A (ZQ0207). The cells were grown at 37 °C in a humidified 5% CO₂ atmosphere. Cell viability was assessed by the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan). Cells were seeded in a 96-well plate with 8000 cells/well, and treated with varying concentrations of COF (0–1.34 µg/mL) for 6 or 24 h. Then, 10 μL CCK-8 solution was well added to each and incubated, at 37 °C, for 1.5 h. An iMark microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, Waltham, MA, USA) was used to measure the absorbance at a wavelength of 450 nm.
BV2 cells were divided into three groups to test whether COF or rapamycin could inhibit microglial activation. After pre-treatment with COF (0.67 µg/mL) or rapamycin (300 nM) (1 × PBS and 1.8 × 10⁻⁴ % DMSO as control separately) for 2 h, LPS (1 μg/mL; Sigma-Aldrich, USA) and IFN-γ (100 ng/mL; Beyotime, Shanghai, China) were added for 18 h. Real-time quantitative PCR was used to test transcripts of pro-inflammatory factors.