The human glioblastoma cell lines U251 and U87 were acquired from the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. DMEM was acquired from GINOM Co., Ltd. (Guangzhou, China). TGX-221 was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO), which was a product purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell viability was assessed using Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. CCK-8 was purchased from Dojindo China Co., Ltd. (Shanghai, China). Approximately 8×10³ cells were seeded in a volume of 100 µl of DMEM into each well of a 96-well plate. TGX-221 was added to the medium and evaluated at different concentrations at a single time-point or at different time-points at a single concentration. In addition, 100 µl of fresh medium containing 5 µl of CCK-8 solution was added into each well and incubated at 37°C for 30 min. The absorbance at 450 nm was measured using a spectrophotometric plate reader. Each group was assessed in triplicate.

The Cell-Light EdU DNA Cell Proliferation kit was purchased from RiboBio Co., Ltd. (Guangzhou, China) and used according to the manufacturer's instructions. Approximately 8×10³ cells were seeded in a volume of 100 µl of DMEM into each well of a 96-well plate. The medium was mixed with TGX-221 at different concentrations, and the cells were evaluated 48 h after exposure to TGX-221. The cells were treated with 50 µmol/l EdU for 12 h at 37°C. After fixation with 4% paraformaldehyde for 15 min, the cells were treated with 0.5% Triton X-100 for 20 min and rinsed with phosphate-buffered saline (PBS) 3 times. Next, the cells were incubated with 100 µl of 1X Apollo^® reaction cocktail for 30 min, and the cell nuclei were stained for 30 min with 5 µg/ml Hoechst 33342. Fluorescence images of the EdU and Hoechst in the cells were captured using a fluorescence microscope (Olympus, Tokyo, Japan). The number of EdU- and DAPI-positive cells was quantified using ImageJ software, and the EdU-labeling index was calculated as the ratio of the number of EdU-positive cells to the number of DAPI-positive cells.

The effects of TGX-221 on apoptosis and cell cycle distribution were determined using the Annexin V-FITC/propidium iodide (PI) apoptosis and cell cycle kit independently, according to the manufacturer's instructions from MultiSciences Biotech (Hangzhou, China). The cells were examined after 48 h following exposure to TGX-221 at different concentrations. At the end of the treatment period, 3×10⁵ or more cells were trypsinized, collected by centrifugation at 1,000 rpm for 5 min and rinsed with cold PBS. Next, the corresponding dyes and solution were added and incubated according to the manufacturer's instructions. Cell apoptosis and cell cycle distribution were analyzed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed using FlowJo software (version 7.6).

Migration and invasion assays were performed using a Transwell chamber with an 8.0-µm pore polycarbonate membrane. The cells were seeded into the top chambers containing the membranes, which were either coated or not with Matrigel for migration and invasion assays, respectively. Then, the chambers were placed into a 24-well plate, and medium containing 10% fetal bovine serum was added. The cells were fixed and stained with crystal violet, which penetrated the underside surfaces of the membranes. Subsequently, the cells were quantified under a microscope. The assays were performed 3 times.

Cell proteins were extracted with RIPA lysis buffer and assessed using the standard BCA method (Beyotime Institute of Biotechnology, Jiangsu, China). Equal amounts of protein were separated using SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked in TBS containing 0.1% Tween-20 and 5% powdered milk, and incubated at 4°C overnight with primary antibodies against cleaved caspase-3, caspase-3, Bcl-2, Lc-3b, MMP9 and β-actin. Then, the membranes were incubated in the secondary antibody Alex Fluor 680/790-labeled goat anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA) for 1 h. Subsequently, the blots were visualized using the LI-COR Odyssey Infrared Imaging System.

All experimental results are expressed as the mean ± SD. A Student's t-test was performed to determine the significance between two mean values. The results were considered significant at P-values of <0.05.

To confirm the effect of TGX-221 on glioblastoma cell proliferation, we performed the CCK-8 assay using different concentrations of TGX-221 in U87 and U251 cells. As shown in Fig. 1A and B, TGX-221 significantly inhibited the viability of U87 and U251 cells in a dose-dependent manner. The IC₅₀ values of TGX-221 in U87 and U251 cells were ~40 and 100 µM, respectively. We then performed the CCK-8 assay at different time-points with the IC₅₀ values of TGX-221. Glioblastoma cell proliferation was inhibited significantly by TGX-221 in a time-dependent manner (Fig. 1C and D).

To further confirm the inhibitory effect of TGX-221 on cell proliferation in glioblastoma cells, we performed the EdU assay in both U87 and U251 cells (Fig. 2A and B). A significant inhibition of cell proliferation was observed in both U87 and U251 cells in a dose-dependent manner. With an increase in TGX-221 concentration, the number of cell nuclei with thymidine analog incorporation was decreased. The total percentage of stained nuclei in cells treated with TGX-221 was lower than that in the cells treated with DMSO (Fig. 2C and D).

In addition, we performed flow cytometry to analyze the cell cycle distribution. As shown in Fig. 3, U87 and U251 cells were cultured with TGX-221 for 48 h. The percentage of cells in the G1 phase was increased compared with that in the control groups. The percentage of cells in the S and G2 phases was decreased, which suggested that TGX-221 inhibited glioblastoma cell proliferation. Importantly, the percentage of S and G2 phases decreased with an increase in TGX-221 concentration in glioblastoma cells.

The effect of TGX-221 on cell apoptosis was investigated using flow cytometry. The apoptosis rates at 48 h after treatment at different concentrations (0,10, 20, 40 and 60 µM) are shown in Fig. 4A and B. We found that the apoptosis rate increased significantly with increasing TGX-221 concentrations.

To examine whether TGX-221 inhibits the migration and invasion of glioblastoma cells, we performed a migration and invasion assay in U87 and U251 cells at different concentrations (0, 10, 20, 40 and 60 µM). We found that TGX-221 inhibited glioblastoma cell migration and invasion (Fig. 5A and B). These results were further confirmed using western blot analyses. Furthermore, we demonstrated that MMP9 gradually decreased with increasing concentrations of TGX-221 (Fig. 5C).