Purified lta

THP1-Xblue-CD14 reporter cells (InvivoGen, France) were cultured and stimulated following manufacturer's instructions. Cells were incubated with 0.1–10 μM FYT21 or 1–40 μM HVF18 and stimulated with 1–1,000 ng ml⁻¹ of P. aeruginosa or E. coli LPS (Sigma), 1 μg ml⁻¹ purified LTA (InvivoGen), 10 μg ml⁻¹ zymosan A (Sigma), 100 ng ml⁻¹ purified flagellin (InvivoGen), 1–100 ng ml⁻¹ recombinant TNF-α (InvivoGen), 0.1% CM of various P. aeruginosa strains or no stimulus. In addition, cells and/or peptide were pretreated with 5–100 μg ml⁻¹ dermatan sulphate (DS36)41 (link). After 18–22 h incubation at 37 °C and 5% CO₂, 20 μl of the supernatants were transferred to new 96-wells plates and 180 μl of QUANTI-Blue (InvivoGen) was added. Plates were incubated at 37 °C and the levels of secreted embryonic alkaline phosphatase (SEAP), which is controlled by the activation of transcription factors NFκB and AP-1, were measured after 1–2 h at an absorbance of 600 nm. To establish the effect of FYT21 on activation of NFκB/AP-1 via TLR9, RAW-Blue reporter cells (InvivoGen) were cultured according to manufacturer's instructions, incubated with 10 μM FYT21 and stimulated with 500 ng ml⁻¹ ODN1826 (InvivoGen).

Cell culture reagents were from PAA. Purified LTA was from Invivogen; LPS, apyrase and reactive blue 2 (RB2), were from Sigma, authentic peroxynitrite was from Cayman, 3-morpholinosydnonimine (SIN-1) was from Invitrogen, MRS2578, MRS2693, and UDP were from Tocris, Amyloid β_1-42 peptide was from EZBiolab, Annexin V was from BioVision. Tumor necrosis factor-α and interleukin-1β Quantikine Elisa Kits were from R&D Systems. NeuN antibody was from Millipore, β-tubulin III antibody was from Sigma, anti-cleaved caspase-3 antibody was from Cell Signaling Technology, Alexa Fluor 488-labeled Griffonia simplicifolia isolectin B₄ and secondary goat-anti-mouse-Alexa Fluor 488 antibody were from Invitrogen, secondary goat anti-mouse-Cy3 and goat anti-rabbit-Cy3 antibodies were from Jackson ImmunoResearch Laboratories. All other materials were purchased from Sigma.