Pi3k inhibitor wortmannin

CGNs and/or CNs were prepared7 (link)8 (link) and seeded at 4 × 10⁵ cells per ml in 96- or 24-well plates with neuronal media for 3 and 21 days, respectively. T-cell lines were re-stimulated with antigen and APCs for 48 h and are referred to as activated T_enc cells. Neurons and activated syngeneic T_enc cells were washed twice and co-cultured at a 1:1 ratio for 24 h, unless stated otherwise. MOG_35–55 T-cell line and MBP_89–101T-cell line were co-cultured with neurons from C57BL6 mice and C57BL/10.RIII mice, respectively.
For some experiments, PI3K inhibitor Wortmannin (catalogue number S2758, Selleckchem), Akt inhibitor MK-2206 (catalogue number S1078, Selleckchem), rIFNβ (12405-1, PBL Assay Science) and anti-PDL1 (10 μg ml⁻¹, Anti-Mouse CD274/B7-H1) Functional Grade Purified, 16-5982-82, eBioscience) were added to the CGNs. Anti-PD1 (10 μg ml⁻¹, Anti-Mouse CD279/PD-1, 16-9985-82, eBioscience) was added to T cells before co-cultures.

HK-2 cells were stimulated with TMAO (300 µM, Sigma-Aldrich, St. Louis, MO, USA), low glucose (5 mM, unstimulated) or high glucose (30 mM, Sigma-Aldrich) for 3 min to 24 h, depending on the experimental setup, at 37 °C in 5% CO₂. The HK-2 cells were also pre-incubated with DMSO (vehicle), Akt inhibitor MK-2206 (1 µM, Selleckchem, TX, USA), mTOR inhibitor ridaforolimus (1 µM, Selleckchem), PI3K inhibitor wortmannin (1 µM, Selleckchem) and ERK inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h prior to TMAO stimulation. Supernatants, cell lysates and total ribonucleic acid (RNA) were collected and kept at –80 °C until further analysis.