Mek1 2 inhibitor pd98059

We performed the transwell migration assay and transwell invasion assay using an 8.0-µm pore size insert (BD Falcon) and a BioCoat^TM Matrigel® Invasion Chamber (Corning, Tewksbury, MA, USA), respectively. To investigate the effects of the coculture, CAF-like cells (5 × 10⁴ cells) in serum-free media were seeded in the lower chambers. The effect of recombinant human PAI-1 (rhPAI-1; R&D Systems) was investigated by addition to the lower chambers. ESCC cells (1 × 10⁵ cells for transwell migration assay; 3 × 10⁵ cells for transwell invasion assay) or macrophages (1 × 10⁵ cells for transwell migration assay; 3 × 10⁵ cells for transwell invasion assay) in the serum-free media were seeded in the upper inserts. After 24 h or 48 h, the cells that migrated through the membranes were stained using Diff-Quik® (Sysmex, Kobe, Japan) and counted. In some experiments, ESCC cells or macrophages were treated with PI3K inhibitor LY294002 (Cell Signaling Technology, Beverly, MA, USA) or MEK1/2 inhibitor PD98059 (Cell Signaling Technology); CAF-like cells were treated with neutralizing antibody against human PAI-1 (AF1786; R&D Systems) or normal goat IgG (AB-108-C; R&D Systems) as the negative control.

Human recombinant IL-1β, TNF-α, and IFN-γ were purchased from Sigma, Missouri, USA. Rabbit polyclonal PDZK1 antibody (PAB15564) was purchased from Abnova, Taipei, Taiwan. NF-κB inhibitor BAY11-7082 and p38MAPK inhibitor BIRB-796 were purchased from Calbiochem (Darmstadt, Germany) and Axon Medchem (Groningen, The Netherlands) respectively. JNK inhibitor SP600125, MEK1/2 inhibitor PD98059 and U0126 were purchased from Cell signaling Technologies, Frankfurt, Germany.

Growth factors: human heregulin-β1 (HRG) was purchased from Proteintech Group, and 17β-Estradiol (E2), Nerve Growth Factor (NGF), Trypsin, N6, Thy1.1, 5-Fluoro-2′-deoxyuridine (FDU), Uridine (U), Forskolin, L-Ascorbic acid, ITS Liquid Media Supplement, Poly-D-lysine Hydrobromide (PDL), 2′-O-Dibutyryladenosine3′,5′-cyclicmonophosphatesodiumsalt (db-cAMP) were from Sigma (St. Louis, MO, United States). Chemicals: MEK 1/2 inhibitor PD98059, phosphoinositide 3-kinase inhibitor LY294002 were from Cell Signaling Technology, E2R inhibitor ICI182780 was from Abcam,andE2Rα inhibitor MPP and E2Rβ inhibitor PHTPP were from Santa Cruz Biotechnology. Antibodies: Chicken anti-myelin protein zero (P0), rabbit anti-phospho-AKT, anti-AKT, anti-phospho-PI3 Kinase, anti-PI3 Kinase, anti-ERK1/2 (ERK), anti-phospho-ERK1/2 were from Abcam, Rabbit anti-myelin associated glycoprotein (MAG), Mouse anti-neurofilament 200 (NF200), anti-β-actin, anti-S100β were from Sigma, and Rabbit anti-ERα and goat anti-ERβ were from Santa Cruz Biotechnology. Other chemicals were obtained from Sigma.