Matrigel

For migration assays, cells were seeded into the upper chamber of a Transwell insert (pore size, 8 μm; Costar) in 100 μL serum-free medium per well. Medium (600 μL) containing 10% serum was placed in the lower chamber to function as a chemoattractant. Nonmigratory cells were removed from the upper chamber by scraping with a cotton bud. The cells remaining on the lower surface of the insert were fixed with 4% formaldehyde (Sigma) and stained by DAPI (Roche). For invasion assays, cells were seeded in a Matrigel (Bio-Rad)-coated chamber and pre-incubated at 37°C for 30 min.

For migration assays, cells were seeded into the upper chamber of a Transwell insert (pore size, 8 μm; Costar) in 100 μl serum-free medium per well. Then, 600 μl medium containing 10% serum was placed in the lower chamber to act as a chemoattractant. Nonmigratory cells were removed from the upper chamber by scraping the membrane. The cells remaining on the lower surface of the insert were fixed with 4% formaldehyde (Sigma) and stained by DAPI (Roche). For invasion assays, cells were seeded in a Matrigel (Bio-Rad)-coated chamber and were incubated at 37°C.

Twenty-four male 4-week old BALB/c-nu mice were procured from the SJA laboratory (Hunan, China). All the animals were housed in an environment with a temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h.
Xenografts were built by subcutaneously injecting 5 × 10⁵ T24 cells into the dorsal flanks; the T24 cells were suspended in 200 mL of a 1:1 mixture of Matrigel (Bio-Rad) and serum-free media. When the tumors had grown to approximately 200 mm³, the 48 mice were randomly divided into four groups (n = 6 for each group): MLN4924 group, celecoxib group, MLN4924+celecoxib group, and DMSO (control) group. The drug solutions of MLN4924 and celecoxib were prepared in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. The MLN4924-treated groups received intraperitoneal injections of 25-mg/kg MLN4924 three times weekly, and celecoxib groups received 50-mg/kg celecoxib orally once a day. All the treatments were given for 4 weeks. The nontreated control group was treated with the same volume of DMSO and drug solution, and the combined group received the same doses of both drugs at the same frequency and duration. Tumor size was measured with a standard caliper every 4 days; the size was calculated using the following formula: length × width2 × 0.5. After 4 weeks of treatment, tumors were excised and photographed.

Human lung fibroblasts (MRC-5) and human LC cells (H1650, A549, H1299, and PC-9) were purchased from the Cell Research Institute of Wuhan University (Wuhan, China) and cultured in RPMI 1640 medium supplemental with 5% fetal bovine serum at a constant temperature. (Hyclone Corporation) Transwell chamber was bought from Millipore, Inc., and matrigel was bought from Bio-Rad (Bio-Rad, Madrid, Spain). Lentivirus kit and retrovirus LV3-PTEN were bought from Shanghai Gemma Biology Co., Ltd.

Human glioma cell lines U-87, C6, U-373, and M059J and human astrocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture conditions were Roswell Park Memorial Institute (RPMI) or Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), at 37°C, 5% CO₂. FBS, RPMI, and DMEM were purchased from Gibco (Rockville, MD, USA). COX-2, extracellular signal-regulated kinase1 (ERK1), phosphorylated (p)-ERK1, c-Jun N-terminal kinase (JNK), and p-JNK were purchased from Abcam (Cambridge, UK). The Transwell chamber was purchased from Corning (Corning, NY, USA). Matrigel was purchased from Bio-Rad (Hercules, CA, USA). Nude mice [6 weeks old, specific pathogen free (SPF)] were provided by the experimental animal center of Sichuan University. TRIzol was purchased from Invitrogen (Carlsbad, CA, USA). Reverse transcription kits (FSQ-101) were purchased from TOYOBO Company (Kyoto, Japan). Lipofectamine 2000 and miR-26b mimic were purchased from Ji Kai Gene Company (Shanghai, P.R. China). Luciferase activity assay kit and luciferase reporter vectors were purchased from Promega (Madison, WI, USA).