Pd98059

For pharmacological treatments 1321N1 cells were plated in a 6-well plate at a density of 1 × 10⁵ cells per well and incubated at 37 °C with 5% CO₂. Compounds (PD98059: 20 µM, Sigma-Aldrich, P215; U0126: 10 nM, Sigma-Aldrich, U120; dissolved in DMSO) were diluted in medium to the final concentration containing 1% DMSO and added to the cultured cells for 48 h (controls contained 1% DMSO as well).
Similarly, PC12 cells were seeded into collagen coated (0.005%, Hoffmann-La Roche) 24-well plates at a density of 3 × 10⁴ cells per well. After 24 h of incubation, the serum was replaced by serum-reduced medium containing medium-diluted pharmacological compounds (K252a: 300 nM, Sigma-Aldrich, 05288; PD98059: 20 µM, Sigma-Aldrich, P215; U0126: 10 nM, Sigma-Aldrich, U120; Bisindolmaleimide: 6 µM, Merck KGaA, Darmstadt, Germany, 203290; LY294002: 50 µM, Sigma-Aldrich, L9908). Then, 6 h later either recombinant NGF (200 ng/mL, SRP3015, Sigma-Aldrich) or medium conditioned by 1321N1 cells and supplemented with the same concentration of the compounds was added.

BBR (Cat# B3251) and PD98059 (Cat# sc-3532) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Santa Cruz Biotechnology (Dallas, TX, USA). The concentration of stocks are 10 mM in water for BBR and 25 mM in DMSO (Sigma-Aldrich, St. Louis, MO, USA) for PD98059.

As IR correlates to glucose intolerance and metabolic disorders [26 (link)], measuring glucose concentrations is important to identify IR. To determine whether the IR models of GC were successfully established, we divided the GCs cultured in 24-well plates into 4 groups. The control group (Con) did not receive any treatment; the Dex group (GD) was treated with Dex (Cisen Pharmaceutical, Jining, China.) (100 μmol/L) for 48 h before harvesting supernatant; the insulin group (GI) was treated with human insulin (Jiangsu Wanbang Biopharmaceuticals, Xuzhou, China) (5 IU) [27 ] for 1 h before harvesting supernatant; the Dex + insulin group (GDI) was treated with Dex (100 μmol/L) for 48 h and insulin (5 IU) was added 1 h prior to the end of the culture. To further investigate the effect of IR either in the absence or presence of inhibition of MAPK signaling pathway on GCs, we divided the GCs cultured in 6-well plates or 24-well plates into 3 groups: Con, GD as described above and the Dex + PD98059 group (GDP) which was treated with Dex (100 μmol/L) for 48 h and PD98059 (Sigma-Aldrich, Steinheim, Germany) at 50 μmol/L was added 4 h before the end of the culture [28 ].