Sml0320

The TLR4 signaling inhibitor CLI-095 (#tlrl-cli95, TAK-242), and the TLR2+4 inhibitor OxPAPC (#tlrl-oxpap1) were purchased from Invivogen (San Diego, USA). ERK 1/2 inhibitor FR180204 (#SML0320), rotenone, dimethylsulfoxid (DMSO), ionomycin and PMA were obtained from Sigma-Aldrich.
rotenone was freshly prepared and dissolved in DMSO prior to the experiments. DMSO concentration in cell culture media did not exceed 0.5%. Argon was purchased in fixed gas mixtures (argon 25, 50 or 75 Vol%, oxygen 21%, respective rest nitrogen) from Air Liquide (Kornwestheim, Germany).

The ADAM8 antibody (B4068) used for Western blotting was purchased from LifeSpan Biosciences. Antibodies against β-actin (AC-15) and β-tubulin (TUB 2.1), and the ERK inhibitor FR180204 (SML0320) were obtained from Sigma-Aldrich. The antibody to detect specific ERK 1/2 phosphorylated forms (pERK1/2) (9101) was obtained from Cell Signaling Technology. The β1-integrin antibody (552828) and its control isotype matched IgG2A (SC3883) were purchased from BD Biosciences and Santa Cruz Biotechnology, respectively. The anti-ADAM8 antibody MAB10311 and its control isotype-matched IgG1 (MAB002) were from R&D Systems.

In order to validate whether HOXB7 truly regulates the migration and proliferation process via AKT/MAPK signaling, three commercially available inhibitors, Akt1/2 kinase inhibitor(A6730, SIGMA, USA), FR180204 (ERK inhibitor II, SML0320, SIGMA, USA) and p38 MAP Kinase Inhibitor IV (SML0543, SIGMA, USA), were choosenfor blocking AKT, ERK and p38α kinase activations, respectively, according the manufacturer's instructions. After MGC-803 cells adhered to the six-well plate, 200 nM of Akt1/2 kinase inhibitor, 200 nM of FR180204 and 130 nM of p38 MAP Kinase Inhibitor IV, were added into the well for blocking the corresponding kinase activation, respectively. After 24h later, the MGC-803 cells were transfected with HOXB7 plasmid, to increase the expression of HOXB7, followed by collected for doing invasion and proliferation assays.