Mek1 2 inhibitor

Dulbecco's modified Eagle medium-F12 (DMEM-F12), and Bovine growth serum (BGS) were purchased from HyClone (South Logan, Utah). Gentamycin, Cisplatin, and Resveratrol were purchased from Sigma-Aldrich (St. Louis, MO). The MEK1/2 inhibitor and the Senescence β-galactosidase staining kit were purchased from Cell Signaling Technology (Danvers, MA). Dead cell apoptosis kit with annexin-V/PI, Trizol reagent and Moloney murine leukemia virus (M-MLV) reverse transcriptase were purchased from Invitrogen (Carlsbad, CA).

HaCaT cells (kindly provided by Dr Toshio Kuroki, Institute of Molecular Oncology, Showa University, Tokyo, Japan) were cultured in Eagle’s minimum essential medium (Sigma-Aldrich, St Louis, MO, USA) containing 10% fetal bovine serum, penicillin G sodium, streptomycin sulfate, and amphotericin B. When 80% confluence was achieved, the cells were trypsinized, washed, and resuspended in the medium at 1 × 10⁶ cells/mL, and 1 mL was added to each well of the 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA). When the cells reached confluence, the medium was completely removed and 1 mL serum-free medium was added to each well. Simultaneously, the recombinant human IL-36β protein (R&D systems, Minneapolis, MN, USA) were added, and the cells were incubated at 37 °C and 5% CO₂. In some experiments, U0126 (MEK1/2 inhibitor; Cell Signaling Technology, Danvers, MA, USA), SB203580 (p38 MAPK inhibitor; Cell Signaling Technology), LY294002 (PI3K inhibitor; Cell Signaling Technology) and sc-514 (IKKβ inhibitor; Abcam, Cambridge, UK), were used. After the indicated time points, cell culture supernatants, and total mRNA were collected for further studies.

Adult human myoblasts were from a young adult [14 (link)], cultured from passage 10 to 14 in Ham's F-10 (Gibco), 10% Bovine Growth Serum (Hyclone), 30 ng/mL FGF2, and 1% penicillin–streptomycin on Matrigel (BD Biosciences) coated plates (1:100 Matrigel:PBS), at 37C and 5% CO2. For experimental conditions involving immunostaining, human cells were plated at 10,000 cells/well in Matrigel coated 8-well chamber slides (1:100 Matrigel: PBS), and cultured for 72 hours with daily re-feedings at 37C in 10% CO2 incubator prior to fixation with 70% ethanol at 4°C. Mouse myoblasts were cultured and expanded in mouse growth medium: Ham's F-10 (Gibco), 20% Bovine Growth Serum (Hyclone), 5 ng/mL FGF2 and 1% penicillin–streptomycin on Matrigel coated plates (1:300 Matrigel: PBS), at 37°C and 5% CO2. For experimental conditions involving immunostaining, mouse cells were plated at 40,000 cells/well on Matrigel coated 8-well chamber slides (1:100 matrigel: PBS) and cultured for 24 hours at 37°C in 10% CO2 incubator prior to fixation with 70% ethanol at 4°C. All experiments using a MEK inhibitor were treated with 10 μM MEK1/2 Inhibitor (U0126, Cell Signaling Technologies).
Human embryonic stem cells (H9 and H7 lines), were cultured in mTeSR-1 (Stem Cell Technologies) as published [17 (link)], and the heparin binding fraction of hESC-Conditioned Medium was prepared as published [17 (link)].