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Advanced Neurology TG100-115 suppresses glioblastoma cell functions

protocols previously described in earlier studies. 13,17 Briefly, (USA), and wound closure was determined as outlined in
U251 cells were cultured in 60 mm dishes with Dulbecco’s prior protocols. 12,13,17
Modified Eagle Medium (Gibco, USA), supplemented
with 10% fetal bovine serum (Gibco, USA) and antibiotics 2.5. Matrigel invasion assay
(100 U/mL penicillin and streptomycin; Gibco, USA), The transwell assay was performed following the
under standard conditions of 5% carbon dioxide and 95% manufacturer’s protocol. To evaluate the invasive capacity
humidity at 37°C. of U251 cells, BioCoat Matrigel invasion chambers
equipped with 8-µm polycarbonate nucleopore filters
2.2. Cell viability and proliferation assay
(Cat. 354480, BD BioSciences, USA) were utilized. After
U251 cells were plated onto 96-well plates at a density of being treated with DMSO (0.1%) or TG100-115 (150 µM)
3,000 cells per well, with 100 µL of culture medium. After for 24 h, 100 µL of cells (2.5 × 10 cells/mL) in serum-free
4
24 h of attachment, the cells were subjected to another 24-h Dulbecco’s Modified Eagle Medium were added to the
treatment with either dimethyl sulfoxide (DMSO; 0.1%, upper compartment of the chamber. As a chemoattractant,
vehicle control) or TG100-115 at various concentrations 600 µL of complete medium was added to the lower
(30, 60, 120, 180, and 240 µM). Subsequently, 10 µL of chamber. Cells that invaded through the Matrigel-coated
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium membrane to the underside of the insert were subsequently
bromide (MTT) reagent (5 mg/mL) was added to each fixed with absolute ethanol and stained using 1% toluidine
well, and the plates were incubated for 3 h at 37°C with blue. Representative fields were imaged under a phase-
5% carbon dioxide. Throughout the incubation period, contrast microscope (CKX41, Olympus, Japan), and the
mitochondrial enzymes converted the yellow MTT into number of invading cells was quantified using NIH ImageJ
insoluble purple formazan. Following this, the medium (USA) via the cell counting module.
was aspirated, and the formazan was dissolved in 100 µL of
DMSO. Cell viability was assessed using a microplate reader 2.6. Western blotting
(Synergy H1, Biotek, USA) by measuring absorbance at
490 nm, which corresponds to the formazan content. The U251 cells were cultured in 35 mm dishes and grown
cell viability was then calculated as a percentage relative to until reaching approximately 60% confluency. Cells were
the control. then treated with either 0.1% DMSO or 150 µM TG100-
115 in fresh culture medium and incubated for 24 h.
2.3. Calculation of half-maximal inhibitory Following treatment, cells were rinsed twice with ice-cold
concentration (IC ) values phosphate-buffered saline to remove the residual serum.
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The half-maximal IC values of TG100-115 following Western blotting analysis was performed as previously
50
12,13,17
24-h incubation in U251 cells were calculated using described. After blocking, the membranes were
non-linear regression analysis in GraphPad Prism 10. incubated overnight at 4°C with primary antibodies: Anti-
Log-transformed concentrations of TG100-115 and the cleaved caspase-3 (1:1,000; 9661S, CST, USA), anti-B-cell
corresponding cell viability data were fitted to a sigmoidal lymphoma 2 (Bcl-2; 1:1,000; 3498S, CST, USA), anti-B-cell
dose-response curve (with variable slope) to determine the lymphoma 2-associated X protein (Bax; 1:1,000; 2772S, CST,
IC value. 24 USA), anti-phosphorylated-Akt (p-Akt; 1:1,000; 9271S,
50
CST, USA), anti-Akt (t-Akt; 1:1,000; 2920S, CST, USA),
2.4. Cell migration assay anti-phosphorylated-ERK1/2 (p-ERK1/2; 1:1,000; 5726S,
Cell migration was measured using a wound healing assay, CST, USA), anti-ERK1/2 (t-ERK1/2; 1:1,000, 4695S, CST,
following established protocols. 12,13,17 Briefly, U251 cells USA), anti-glyceraldehyde-3-phosphate dehydrogenase
were cultured in 24-well plates at a density of 1 × 10 /mL (1:5,000; 2118S, CST, USA), anti-phosphorylated-
5
per well. A monolayer of cells was scratched with a sterile cofilin (p-cofilin; 1:1,000; ab12866, Abcam, USA), anti-
200 µL pipette tip to create a wound gap. After rinsing the cofilin (t-cofilin;, 1:1,000; ab134963, Abcam, USA), and
wells with phosphate-buffered saline to remove detached anti-TRPM7 (1:1,000; ab85016, Abcam, USA). On the
cells, cultures were treated with either DMSO (0.1%) following day, mouse (1:5,000; 7076S, CST, USA) or rabbit
or TG100-115 at various time points (24, 48, and 72 h) (1:10,000; 7074S, CST, USA) secondary antibodies were
or various concentrations. For each well, images from applied, respectively. Protein bands were detected using
four different fields were captured using a digital camera a chemiluminescence reagent system (iBright Imaging
connected to an Olympus phase-contrast microscope (×10 System FL1500, Thermo Fisher Scientific, USA). Band
objective; CKX41, Olympus, Japan). The wound gap was intensities were quantified using NIH ImageJ (USA) for
quantified using the wound healing tool in NIH ImageJ densitometric analysis.

Volume 4 Issue 3 (2025) 90 doi: 10.36922/AN025110023