2.1. Cell source and processing

The HL-1, 3T3-L1, and RAW264.7 cell lines were purchased from the Chinese Academy of Medical Sciences and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) in a 37°C, 5% CO₂ incubator before the experiment.

Adipocyte differentiation was induced as previously described (12). The cells were cultured in 6-well plates (5×10⁵ cells per well) and transfected. The expression of ADPN or STAT3 in adipocytes was knocked down using 9 μL of lipo2000 (11668-019, Invitrogen), 50 nM of the appropriate siRNA 3 μL [siADPN pool (mmu-ADPN-1, mmu-ADPN-2, and mmu-ADPN-3; RIBIBIO, China) or siSTAT3 pool (mmu-STAT3-1, mmu-STAT3-2, and mmu-STAT3-3; RIBIBIO, China)], and 250 μL of OptiMEM (31985-070, Gibco) per well. STAT3 was overexpressed in adipocytes using lipo2000, STAT3 plasmid (RIBIBIO, China), and OptiMEM. Non-sense sequences were used as negative controls (210011, Ubigene).

RAW 264.7 macrophages with HMGB1 knockdown were obtained using adenovirus (m-HMGB1-shRNA-GFP-Puro from 293T cells, 6.57 × 10⁸ TU/mL, MOI:100, 7.6 L, Genechem, Shanghai). Cells were cultured in 6-well plates (5 × 10⁵ cells per well), transfected using shRNA HMBG1 (MOI = 10), and screened using puromycin (2 μg/mL, Genechem, Shanghai) to obtain the infected RAW 264.7 macrophages.

RAW 264.7 macrophages were maintained in high-glucose DMEM (Gibco, USA) containing 10% fetal bovine serum (AusgeneX, Australia) and 1% penicillin/streptomycin (Solarbio, China) at 37°C under normal (5% CO₂ and 95% air) or hypoxia (5% CO₂, 94% N₂, and 1% O₂) conditions. To determine the effects of adipocyte-derived ADPN on macrophages, the cells were cultured in the conditioned medium (CM) from siNC or siADPN adipocytes. To determine the macrophage receptor that binds adipocyte-derived ADPN, macrophage receptor-binding antibodies, including AdipoR1 (ab126611), AdipoR2 (ab77612), aVb3 (EMD Millipore, MAB1876-Z), and aVb5 (EMD Millipore, MAB1961), were added to siNC CM, which was then administered to macrophages.