Anti il 8

The human promonocytic cell line U937 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Life Technologies, Monza, Italy), 100 U mL^-1 penicillin, 100 μg mL^-1 streptomycin, and 2 mm glutamine (Sigma-Aldrich, Milan, Italy) at 37 °C with 5% CO₂. The cells were dispensed at 1 × 10⁶/mL and made quiescent through overnight incubation in serum-free medium. They were then placed in RPMI 1640 medium with 2% fetal bovine serum and treated with 6 μm 27-OH (Avanti Polarlipids, Alabaster, AL, USA) or in serum-free RPMI 1640 medium and treated with 5 μm HNE (Alexis, Vinci-Biochem, Vinci, Italy). In certain experiments, cells were co-treated with anti-TLR4 (0.2 μg mL^-1), anti-IL-8 (0.04 μg mL^-1), anti-IL-1β (0.04 μg mL^-1), or anti-TNF-α (0.04 μg mL^-1) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In others cases, cells were pretreated for 1 h with 1 μm parthenolide (PTN) (Sigma-Aldrich), a specific inhibitor of NF-κB nuclear translocation. Final concentrations and incubation times for all experiments are reported in the figure legends.

After dewaxing, hydration, and antigen retrieval, the IgAN kidney section samples were incubated with anti-caspase-3, anti-caspase-9, anti-TGF-β1, anti-CoI-IV, anti-Fibronectin-1, anti-IL-1β, anti-IL-6, and anti-IL-8 (Santa Cruz Biotechnology Inc, Santa Cruz, CA) overnight at 4 °C. Then, samples were incubated with biotin-labeled secondary antibody (Abcam, Cambridge, UK, 1:10,000) for 30 min at room temperature. Images were captured using light microscopy.

A549 and H292 cells (1 × 10⁶ cells/well) were seeded in six-well plates overnight and were then exposed to the different treatments at the indicated time points. Cell lysates and tumour tissue homogenates were separated by SDS–PAGE and then transferred onto PVDF membranes (Millipore, USA). Next, WB was performed as previously described23 (link). The following specific primary antibodies were used: anti-phospho-STAT1 (1:1000, Tyr701, CST#9167), anti-STAT1 (1:1000, Tyr701, CST#9167), anti-IL-6 (1:1000, 21865-1-AP), anti-IL-8 (1:1000, sc-1265; Santa Cruz Biotechnology), anti-VEGF (1:1000, BA0407; Boster, China), and anti-GAPDH (1:5000, BL1039; Wuhan, China).