Interleukin 6 (il 6)

For cell surface markers, cell pellets were incubated with ice RIPA buffer (Thermo-Fisher, Cat# 89901, Massachusetts, USA) for 15 min, then centrifuged at 14,000 rpm for 20 min. Supernatant containing cell lysates were analyzed using ELISA kits: iNOS (LS-Bio; Washington, USA, Cat# LS-F39142), CD206 (Sigma-Aldrich; Cat# RAB1178, Missouri, USA) following manufacturer’s instructions. Cell culture supernatants were used to measure cytokine levels: IL-6 (LS-Bio; Cat# LS-F9754, Washington, USA), TNF-α (LS-Bio; Cat# EH3TNFA, Washington, USA), and IL-10 (Sigma-Aldrich; Cat# KHC0101, Missouri, USA). All ELISA experiments were done in triplicates.

The cultured cells were lysed in RIPA buffer plus protease inhibitor (PMSF). Total cellular protein was collected by centrifugation, and the concentration determined by the BCA method. Proteins were first separated by SDS-PAGE and then transferred to PVDF membranes (0.45 µm, Millipore). The PVDF membranes were washed with TBST, blocked for 1 h with 5% skim milk powder dissolved in TBST, and incubated with primary antibodies against TGF-β (Calbiochem, MA, USA), Smad3 (Santa Cruz, CA, USA), p-Smad (Santa Cruz, CA, USA), Collagen I (Merck Millipore, MA, USA), C3a (Abcam, MA, USA), C3aR (GeneTex Inc., USA), IL-6 (LSBio, Seattle, WA, USA), p-IKBα (Abcam, MA, USA) and IKBα (Santa Cruz, CA, USA) at 4°C overnight with dilutions recommended by the manufacturer. β-actin was used as an internal reference. The PVDF membranes were washed with TBST and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at 37°C for 1 h. Protein bands were detected using chemiluminescence (ECL) reagent (Pierce, Thermo Scientific). The quantitative analysis of protein band density was performed on Quantity One (Bio-Rad).