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Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01 Page 5 of 25

China). The environment for cultured cells needs to meet three conditions simultaneously: water saturation,
a temperature of 37 °C, and 5% CO in the air. IsocuB, with a purity of ≥ 99.0%, was obtained from Chengdu
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Must Bio-technology Co., Ltd., China. TMZ was purchased from MCE in China with a purity of ≥ 99.0%.
The drugs were dissolved in dimethyl sulfoxide (DMSO). The DMSO was diluted to less than 0.1% to
ensure that it had no effect on the cells.

Reagents
CCK-8 (Dojindo, Japan), Matrigel Matrix (BD Biosciences, USA), paraformaldehyde (Solarbio, China),
crystal violet (Sigma, USA), BeyoECL Plus (PIERCE, USA), TUNEL Assay Kit (Yesen, China), Annexin v-
FITC/PI Apoptosis Kit (Boster, China), TRIzol Solution (Invitrogen, USA), Reverse Transcription System
(Promega, USA), DEPC (Sigma, Germany), GoTaq® qPCR Master Mix (Promega, USA), Primers (Sangon
Biotech, China), Bovine Serum Albumin (BSA) and BAC Protein Quantifier Kit (Beyotime Biotechnology,
China), N-cadherin, Vimentin, BCl-2, Hsp90, p38-MAPK, p-MAPK, MMP-2, MMP-9, PDK1, anti-AKT,
anti-p-AKT (CST, USA), p-STAT3 and STAT3 (Abclonal, China), β-actin (Servicebio, China), Bulge-Loop
miRNA ORT-PCR Starter Kit, Human Bulge-Loop hsa-miR-1268a Primer Set, Bulge-Loop U6 gPCR
Primer Set, Human microFF hsa-miR-1286 Inhibitor and microFF Inhibitor NC (RIBOBIO, China),
micrON hsa-miR-1286 mimic (Genepharma, China) were used in this study.

Cell proliferation assay
We evaluated the effect of isocuB on glioma cell proliferation using a CCK-8 assay. We initially used 0.25%
trypsin to digest U251 and U87 cells at the logarithmic growth stage and subsequently diluted them to a
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seeding density of 7 × 10 cells/mL in 96-well plates. IsocuB was then diluted to seven concentrations (0.001,
0.01, 0.1, 0.5, 1, 5, and 25 µmol/L). TMZ was then diluted to seven concentrations (10, 50, 100, 200, 500,
1,000, and 2,000 µmol/L). These were added 12 and 24 h later. Finally, the CCK-8 reagent was added at 12
and 24 h for absorbance detection at 450 nm using a microplate reader. Triplicate experiments were
performed independently {Cell viability = [(OD Experiment group - OD Blank )/(OD Control group - OD Blank )] × 100%}.

Wound healing assay
We assessed the impact of isocuB on glioma cell migration by conducting a wound-healing assay. We
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diluted the cell density of U251 to 2.5 × 10 cells/mL and plated it into a 6-well plate. Five parallel lines
(2 mL per well) were drawn beside each well with a marker. Then, 24 h later, the lines were drawn with a
200 µL pipette tip tilted at a 45° angle, washed three times with PBS, and DMEM dilution containing 1%
FBS in three concentrations of isocuB (0, 0.01, and 0.1 µmol/L) was added. Finally, five visual fields were
randomly selected at 12, 24, and 36 h to capture images and observe the migration of U251 cells. Triplicate
experiments were performed independently.

Transwell invasion
To assess the invasive impact of isocuB on glioma cells, a transwell invasion assay was conducted. The
diluted matrix adhesive was added to the upper chamber and placed in an incubator for 8 h to allow the
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matrix to form and absorb excess fluid. After 12 h of cell starvation, 5 × 10 cells were seeded into the upper
chamber with 100 μL of 0.2% BSA DMEM containing isocuB (0, 0.01, and 0.1 µmol/L), and 500 μL of
complete DMEM medium was added to the lower chamber. After 24 h, non-migrated cells on the filter side
of the upper chamber were removed using a cotton swab, fixed with 4% paraformaldehyde for 1 h, rinsed
three times with PBS, and stained with 1 mL of crystal violet for 1 h. The transwell membrane was then
covered with a cover glass, and the migrated cells were counted under a microscope. Triplicate experiments
were performed independently.