Opti memtm medium

The day before knockdown or overexpression, cells were seeded in a six-well plate to achieve a cell density of 40%. The culture medium was then removed, and the cells were washed twice with sterile PBS. Each well of the six-well plate was filled with 1.5 mL of Opti-MEMTM medium (Gibco, USA). Additionally, a sterile centrifuge tube was prepared, and 0.5 mL of Opti-MEMTM medium, 5μL of small interfering RNA (siRNA) or overexpression plasmid, and 5μL of Lipofectamine 2000 (Thermo Fisher Scientific, Shanghai, China) were mixed and allowed to stand for 20 min. The mixture was then added to the experimental group cells, while the control group received the same mixture without siRNA or overexpression plasmid. After 6 h, the medium was replaced with complete culture medium, and the cells were cultured for 48 h before collection for analysis.

Maxisorp NUNC-immuno plates were coated with streptavidin (from Streptomyces avdinii, Sigma) at 1 µg/well in PBS and incubated at 4 °C overnight. All incubations were performed in a humid chamber. To avoid unspecific binding, the plates were blocked with PBS containing 1% BSA (Sigma-Aldrich) for 1 h at 37 °C. After the blocking procedure, the plates were washed three times with pre-warmed PBS.
To investigate the interactions of Pam2 with human 18S rRNA and TLR2 fusion protein, Pam2-Biotin (Pam2-Biotin-Aca-Aca-NH2, Genaxxon bioscience) was preincubated with human 18S rRNA in ultra-pure water (10 µL) for 30 min at 37 °C. Subsequently, TLR2 fusion protein and medium were added (pure Opti-MEMTM medium, Gibco) for an additional 30 min at 37 °C. This mixture was added to the streptavidin-coated plates (50 µL/well) and incubated for 15 min at 37 °C, followed by three washing steps with PBS.
For the detection of TLR2, each well was incubated with an anti-human IgG-peroxidase conjugated antibody (1:1000, Dako) for 1 h at 37 °C. After the last washing step, a substrate buffer with 20 mg o-phenylenediamine dihydrochloride (OPD, Sigma) and 30% of H₂ O₂ was applied to the wells (50 µL) and incubated for approximately 20 min at room temperature in the dark. The reaction was stopped by adding 25 µL/well 2 M H₂ SO₄, and absorption was measured with a photometer at 450–650 nm

Transfection was performed using Opti-MEM^TM medium (Gibco^TM, Carlsbad, CA, USA) and the Lipofectamine 3000 Transfection Kit from Invitrogen (Carlsbad, CA, USA) for 20000 cells (TeloHAEC) in a 96-well format.
COX-2 promoter activity was measured with pDRIVE5-Lucia-hCOX2 (InvivoGen, Waltham, MA, USA) controlling the expression of a secreted luciferase enzyme and pSELECT-zeo-SEAP (InvivoGen) as a control plasmid to normalize the transfection efficiency. Both plasmids were used at a 1:1 proportion, and their activity was measured using QUANTI-LucGoldTM (InvivoGen) and QUANTI-Blue (InvivoGen) kits, respectively.