Transwell chambers were incubated for 24?h
Transwell chambers were incubated for 24?h. epidermal growth factor (EGF; Thermo Fisher Scientific, USA), and 10?ng/mL basic fibroblast growth factor (bFGF; PeproTech, USA). GBM and NHA cells were cultured in Dulbeccos altered Eagles medium (DMEM; Life Technologies-Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies-Thermo Fisher Scientific, USA) and maintained at 37?C in a humidified chamber containing 5% CO2. Recombinant human PDGF-AA (Peprotech, USA) was dissolved in phosphate buffered saline (PBS), and AG-1296, an inhibitor of PDGFR (Selleck, China), was dissolved in DMSO before addition to media. The small molecule MK-2206 (Apexbio, USA) was dissolved in DMSO Rigosertib and used as an inhibitor of AKT phosphorylation. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from the cells using Trizol reagent (Takara, Japan) according to the manufacturers protocol. Total RNA (1?g) was reverse-transcribed, and the resulting cDNA was used as a template in qRT-PCR using a standard SYBR premix Ex Taq (Takara, Japan) around the Real-Time PCR Detection System (Roche, 480II, USA). served as the internal control, and experiments were conducted in triplicate. The following primers Rigosertib were used: forward, 5-AATGAAGGGGTCATTGATGG-3, reverse, 5-AAGGTGAAGGTCGGAGTCAA-3; forward 5-CCGGAGCCTCGAAAAGAGATT-3, reverse 5-ATGATCCGTGTCTGGAGGTC-3. Western blotting analysis Cells and tissues were lysed in RIPA C14orf111 buffer (Pierce-Thermo Fisher Scientific, USA) made up of a protein inhibitor cocktail. Protein concentrations were quantified using Pierce Protein Assay Kit (Pierce, USA). Proteins (20?g) were separated by SDS-PAGE, and detected by primary antibodies for GOLM1(1:500; Abcam), GSK3 (phospho S9) (1:5000; Abcam), GSK3 (1:2000; Abcam), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; Cell Signaling Technology; Danvers, MA, USA), p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling Technology), phospho-AKT (Ser473) (1:1000; Cell Signaling Technology), Snail (1:1000; Abcam), ZEB1(1:1000; Abcam), AKT (pan) (1:1000; Cell Signaling Technology), phospho-PDGF receptor (Tyr754) (1:1000; Cell Signaling Technology) and GAPDH (1:2000; Cell Signaling Technology). Proteins were quantified using chemiluminescence (Bio-Rad, USA) according to the manufacturers protocol. Construction of stably transfected cells Lentiviral constructs made up of full length GOLM1 (Lenti-GOLM1; GeneChem Technologies; Shanghai, China) or short hairpin RNAs (sh-GOLM1C1, sh-GOLM1C2; GeneChem Technologies) were used to generate stable GOLM1 overexpressing or knockdown cell lines. U251, A172 and P3#GBM cell lines were infected with sh-GOLM1C2, while U87MG cells were infected with Lenti-GOLM1. After 48?h, U87MG, U251, A172 and P3#GBM cells were exposed to puromycin (0.5?g/mL, 2?g/mL, 2?g/mL and 2?g/mL respectively; A1113802; Gibco-Thermo Fisher Scientific) for an additional 2?weeks to enrich for cells harboring the constructs. The targeting sequences in Rigosertib the shRNAs were the following: sh-NC 5-TTCTCCGAACGTGTCACGTtt-3; sh-GOLM1C1 5- GTGGCTTAGAATTTGAACAtt-3; sh-GOLM1C2 5- CAAGCTGTACCAGGACGAAtt-3. Migration and invasion Rigosertib assays. Invasion and migration of U87MG, U251 and A172 cells were evaluated in uncoated and matrigel-coated (BD Biosciences; Bedford, MA, USA) Transwell chambers (8?m pores; Corning Costar; Oneonta, NY, USA). Cells (2??104) were seeded in the top chamber in DMEM (200?L) with 1% FBS and the lower chamber was filled with DMEM (600?L) containing 30% FBS. Transwell chambers were incubated for 24?h. Cells that had invaded or migrated into the lower surface were fixed with 4% paraformaldehyde (Solarbio; Beijing, China), stained with crystal violet (Solarbio) for 20?min, and counted under bright field microscopy. Rigosertib Images were acquired from 5 random fields in each well, and cell numbers were decided using Kodak MI software. Each experiment was performed in triplicate. For 3D spheroid invasion assay, spheroids were generated through incubating P3#GBM cells in the spheroid formation matrix for 96?h in a 3D culture qualified 96-well spheroid formation plate. Spheroids were embedded into the invasion matrix (Trevigen, USA) composed of basement membrane proteins in the 96-well plate. Glioma.