Human glioma cell lines U87MG and U373MG (Korean Cell Line Bank (KCLB), Seoul, Korea), and an immortalized primary human fetal astrocyte cell line IM-PHFA were previously described (36,37). The Lenti-X 293T cell line for lentivirus packaging was purchased from Clontech (Mountain View, CA, USA). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml of antibiotic-antimycotic (both from Lonza, Walkersville, MD, USA) at 37°C in a humidified incubator with 5% CO₂, and the viability of the cultured cells was monitored by LUNA-FL Automated Cell Counter (Logos Biosystems, Gyeonggi-do, Korea).

4′,6-Diamidino-2-phenylindole (DAPI) and rhodamine-phalloidin were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). G418 and puromycin were purchased from Biomax (Seoul, Korea). Polybrene was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Y-27632 was purchased from Sigma-Aldrich. The antibodies used were purchased as following: TCF8/ZEB1, Slug, E-cadherin, N-cadherin, ZO-1, RhoA, Cdc42, Rac1/2/3, phospho-Rac1/Cdc42, and active Rho detection kit from Cell Signaling Technology (Danvers, MA, USA), and β-actin from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Lentiviral vectors expressing AEG-1 shRNA were purchased from Sigma-Aldrich (SHCLNG-NM_178812). Control viral vector pLKO.1 GFP shRNA plasmid (Addgene; plasmid #30323) was a gift from Dr David Sabatini (Massachusetts Institute of Technology, Cambridge, MA, USA), and the envelope vector pMD2.G (plasmid #12259) and packaging plasmid psPAX2 (plasmid #12260; both from Addgene) were kindly provided by Dr Didier Trono (Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland). The lentiviral vector expressing AEG-1 was purchased from OriGene (RC207238L1). Each lentivirus expressing AEG-1 (lenti-AEG-1), AEG-1 shRNA (lenti-AEG-1sh) or GFP shRNA (lenti-GFPsh) was produced by co-transfection with each expression plasmid, pMD2.G and psPAX2 into Lenti-X 293T cells. Transfections were carried out using Lipofector-2000 (AptaBio, Gyeonggi-do, Korea) according to the manufacturer's instructions. Media were harvested at 48, 72 and 96 h post-transfection and the media were centrifuged at 1,500 rpm for 5 min and filtered using a 0.45 µm syringe filter for removing inadvertently collected cells. Every infection was carried out in the presence of 8 µg/ml of Polybrene. After transduction, cells were selected with 2 µg/ml puromycin.

Whole cell lysates were prepared, and western blot analysis was performed as previously described (38). Active Rho detection was performed with an active Rho detection kit (Cell Signaling Technology; #8820) according to the manufacturer's protocol. Seven hundred micrograms of total proteins were used to pull down active Rho with GST-RBD of Rhotekin. Primary antibodies for TCF8/ZEB1 (1:1,000), Slug (1:1,000), ZO-1 (1:1,000), N-cadherin (1:1,000), E-cadherin (1:1,000), RhoA (1:1,000), Rac1/2/3 (1:1,000), Cdc42 (1:1,000), phospho-Rac1/Cdc42 (1:1,000), active-Rho (1:1,000) and β-actin (1:10,000) were used for immunoblotting followed by horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:5,000) for 1 h, and visualized using the enhanced chemiluminescence detection system.

Cells were plated in 8-well chamber slides and incubated for 24 h in complete medium. Then the cells were fixed with 4% paraformaldehyde in PBS for 30 min and the slides were incubated with 0.1% Triton X-100 in PBS for 5 min for permeabilization and blocked with 1% BSA in PBS for 20 min. Then the cells were incubated with rhodamine-phalloidin for 20 min and DAPI for 1 min at room temperature. Images were taken with a FluoView FV1000 confocal microscope (Olympus) and iRiS™ Digital Cell Imaging System (Logos Biosystems).

In vitro invasion assays were performed using 48-well Boyden chambers (Nuero Probe, Inc., Gaithersburg, MD, USA). A polycarbonate membrane with 8-µm pore size (Nuero Probe) was coated with Matrigel and dried overnight. The Boyden chambers were filled with medium containing 10% FBS in the lower compartment, and the coated membrane was mounted in the chamber. Fifty microliters of U87MG cells in serum-free media were placed in the upper chamber of the apparatus and allowed to settle onto the Matrigel-coated membrane. Boyden chambers were incubated at 37°C in a CO₂ incubator for 16 h. After incubation, the membranes were removed, fixed, and stained with Diff-Quick staining kit. Non-motile cells were removed with cotton swabs from the upper surface of the membrane. Motile cells on the bottom face of the membrane were photographed and the stained cells were counted in three randomly chosen fields using a microscope.

U373MG cells were seeded into 6-well plates and incubated for 24 h until reaching 80–90% confluency. A 200-µl pipette tip was used to make scratches in each well. Several regions were marked and photographed at 0 and 24 h after the scratches were made. Phase-contrast microscopy images were taken using Zeiss Axio. Cell motility was calculated as the area covered by the cells between the edges at the time of measurement. Migration rate = (mean area occupied by cells/mean original area) × 100. Each test group was assayed in triplicate at least.

Data are presented as the mean ± standard error of the mean (SEM) and were analyzed for statistical significance using the unpaired Student's t-test. P<0.05 was considered statistically significant.

To examine whether AEG-1 promotes mesenchymal transition of glioblastoma cells, we modulated AEG-1 expression in normal astrocytes (IM-PHFA), and glioblastoma cells (U373MG and U87MG) (Fig. 1A and C). Overexpression of AEG-1 in IM-PHFA cells intensively induced expression levels of the mesenchymal markers ZEB1, Slug and N-cadherin, but reduced expression of the epithelial marker ZO-1 (Fig. 1B). Consistently knockdown of AEG-1 in human glioblastoma cells decreased expression of mesenchymal markers ZEB1, Slug and N-cadherin, but increased expression of epithelial markers E-cadherin and ZO-1 (Fig. 1D). These results indicate that AEG-1 may promote mesenchymal transition of human glioblastoma cells.

Since mesenchymal transition of glioblastoma is involved in the invasion and migration of cancer cells, we then aimed to ascertain whether AEG-1 affects invasive ability and motility of human glioblastoma cells. As shown in Fig. 2A and B, knockdown of AEG-1 in glioblastoma cells decreased more than 58% of invaded cells compared with the control. In addition, wound healing assays showed that knockdown of AEG-1 significantly inhibited the migration of glioblastoma cells (Fig. 2C and D). Collectively, these results suggest that AEG-1 may play an important role in the invasion and migration of glioblastoma cells through the induction of mesenchymal transition.

Actin stress fibers that induce cytoskeleton remodeling are associated with cellular contractility providing force for increased cell movement during mesenchymal transition (39). Thus, we examined whether AEG-1 modulates stress fiber formation. Overexpression of AEG-1 in IM-PHFA cells significantly increased actin stress fibers (Fig. 3A). In accordance, knockdown of AEG1 in glioblastoma cells dramatically decreased stress fibers (Fig. 3B). These results indicate that AEG-1 increases actin stress fiber formation to induce changes in actin cytoskeleton structures and the motility of human glioblastoma cells.

During mesenchymal transition, cells require dynamic actin rearrangement and cytoskeleton remodeling via regulation of Rho family GTPases (39). Overexpression of AEG-1 in IM-PHFA cells intensively increased the active form of RhoA (RhoA-GTP) and phosphorylation of Rac1 and Cdc42, and also increased the protein levels of the Rho family GTPases (Fig. 4A). Knockdown of AEG-1 in glioblastoma cells significantly reduced expression of the Rho family proteins as well as their activation (Fig. 4A). Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) is a major downstream protein of RhoA, which promotes F-actin stabilization and induction of Rac signaling (39). To confirm whether AEG-1-induced RhoA activation is critical for mesenchymal transition, we used Y-27632 a specific inhibitor of ROCK1. As shown in Fig. 4B, treatment with Y-27632 attenuated AEG-1-induced stress fibers. Collectively these results revealed that AEG-1 activates Rho signals to induce stress fiber formation and mesenchymal transition.