Ap 1 inhibitor sr11302

Bladder cancer T24 cells were obtained from the ATCC (Manassas, VA, USA) and cultured in DMEM (Logan, UT, USA) mixed with 0.6% penicillin-streptomycin and 10% FBS in 5% CO₂ at 37 °C. When the confluence reached 50–60%, the cells were treated with different concentrations of LPA (Sigma-Aldrich, St. Louis, MO, USA). To investigate the effects of LPA on RON expression in T24 cells, the cells were collected at various times, and the RON expression levels were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses. The roles of specific signal transduction pathways in LPA induced RON expression were determined by pretreatment of T24 cells with the specific MAPK/Erk kinase (MEK) inhibitor PD98059 (NEB, Beverly, MA), p38 MAPK inhibitor SB203580 (Calbiochem, La Jolla, CA, USA), JNK inhibitor SP600125 (Calbiochem, La Jolla, CA, USA), NF-κB inhibitor BAY11-7082 (Calbiochem, La Jolla, CA, USA), and AP-1 inhibitor SR11302 (Tocris Bioscience, Ellisville, MO, USA) for 1 h prior to exposure to LPA.

All agonists, drugs and inhibitors were dissolved in glycerol, DMSO or water. Sodium salt of SCFAs were from Sigma and used in a range of concentrations from 0.5 to 8 mM. GPRs agonists: GPR41: 4-CMTB (1 μM Tocris) and Tiglic acid (1–10 mM Sigma); GPR43: AR420626 (1 μM Cayman) and 1-MCPC (1mM Sigma); GPR109a: Niacine (1–10 mM, Sigma) and MK1903 (1 μM Tocris). GPRs sub-unit inhibitors used were: Pertussis toxin (Ptx 0.2 μg/ml) and U73122 (10 μM) from Sigma. HDAC inhibitors: Trichostatin A (TSA 1 μM Sigma), SAHA (5 μM Sigma) and valproic acid (VPA 5 mM Sigma). SP1 inhibitor Mithramycin A (0.1 μM Sigma). PPARγ activators: Pioglitazone (5 μM), Roziglitazone (10 μM) and PPARγ inhibitor G9662 (100 μM), from Cayman. NF-kB inhibitor BAY 11-7082 (40 μM). AP-1 inhibitor SR-11302 (10 μM Tocris). STAT3/Jak2 inhibitor Cucurbitacin I (1 μM) from Tocris. IFNγ (100 U/ml) and TNFα (10 ng/ml) were from Peprotech. Final concentration of DMSO had no detectable effect on cells viability or responses.