Mtor inhibitor ridaforolimus

Renal fibroblasts were stimulated with TMAO (300 µM, Sigma-Aldrich, St. Louis, MO, USA), TNF-α (1, 10 or 50 ng/ml, Sigma-Aldrich) or the combination of both for 24-96 h, depending on the experimental setup, at 37 °C in 5% CO₂. Renal fibroblasts were pre-stimulated with TMAO for 2 h prior to TNF-α stimulation during the combination treatments. The renal fibroblasts were also pre-incubated with DMSO (vehicle), mTOR inhibitor ridaforolimus (1 µM, Selleckchem, TX, USA), Akt inhibitor MK-2206 (1 µM, Selleckchem), PI3K inhibitor wortmannin (1 µM, Selleckchem) or ERK inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany) for 1 h prior to TMAO or TNF-α stimulation. Supernatants were collected and stored at -80ºC until further investigation.

Renal fibroblasts were stimulated with TMAO (100 µM, 200 µM, or 300 µM; Sigma-Aldrich) for 24–96 h, depending on the experimental setup, at 37 °C in 5% CO₂. As a positive control, renal fibroblasts were stimulated with TGF-β1 (10 ng/mL Invivogen, CA, USA). The renal fibroblasts were also pre-incubated with DMSO (vehicle), PERK inhibitor GSK2656157 (0.5 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), Akt inhibitor MK-2206 (1 µM, Selleckchem, Houston, TX, USA), mTOR inhibitor ridaforolimus (1 µM, Selleckchem), and PI3K inhibitor wortmannin (1 µM, Selleckchem) for 1 h prior to TMAO stimulation. Supernatants were collected and kept at −80 °C until further analysis.