Dmed f12 medium

Primary microglia were isolated from brains of neonatal C57BL/6 mice (P0–P3) as described.²¹ The purified microglial cells were cultured at 37°C in DMED–F12 medium (Gibco) containing 10% fetal bovine serum (Gibco). After 7 days, microglia were pre‐treated with 10, 50, or 100 μM ASD (Alfabiotech) or pioglitazone (10 μM, Sigma‐Aldrich).³⁷ After 30 min, microglia were treated for 24 or 48 h with either 50 ng/mL LPS (Sigma‐Aldrich) or phosphate‐buffered saline (PBS; BOSTER). Experimental groups were as follows: control group (Ctrl), not treated with ASD or LPS; LPS group (LPS), treated with LPS but not ASD; LPS + ASD (10, 50, 100 μM) group, treated with LPS and ASD at the indicated concentrations; and LPS + pioglitazone group, treated with LPS and 10 μM pioglitazone. At each time point, microglia were collected and transferred to new plates for further experiments.

Cells were transfected with siRNAs-Tlr2, siRNA-Beclin1 or with control siRNA using Lipofectamine® 3000 (Lipo3000, Thermo Fisher Scientific, Inc., MA, USA) at 37 °C in a humidified incubator with 5% CO₂. The siRNAs were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Another group of cells was transfected with a GFP-labeled nonspecific siRNA that served as the negative control (NC). The sequences of the siRNAs used in the present study were as follows: siRNA-Tlr2 sense: 5′-CAG AUC UAC AGA GCU AUG ATT-3′, anti-sense: 5′-UCA UAG CUC UGU AGA UCU GTT-3′; siRNA-Beclin1 sense: 5′-GUG GAA UGG AAU GAG AUU ATT-3′, anti-sense: 5′-UAA UCU CAU UCC AUU CCA CTT-3′; NC-siRNA sense: 5′-UUC UCC GAA CGU GUC ACG UTT-3′, anti-sense: 5′-ACG UGA CAC GUU CG GAG AAT T -3′. When NPCs seeded into 6-well plates reached 80% confluence, transfection was performed by mixing 5 µL siRNA with 5 µL Lipo3000 in a final volume of 2000 µL DMED/F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 15% serum without antibiotics, according to the manufacturer’s protocol. After 16 h of transfection, the cells were infected with P. acnes for 8 h. Finally, the mRNA and protein were extracted from the cells. Transfections were performed in triplicate, and the experiment was repeated at least three times.