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Page 6 of 25 Han et al. Cancer Drug Resist 2024;7:16 https://dx.doi.org/10.20517/cdr.2024.01

TUNEL staining
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The U251 cells in the growth stage were seeded in 6-well plates at a density of 2.5 × 10 cells/mL, with 2 mL
per well. The drug (0, 0.1, 0.5, and 1 µmol/L) was added after cell adhesion. After a 24-hour incubation, the
cells were harvested using ethylenediaminetetraacetic acid-free pancreatic enzymes and washed twice with
PBS. Each sample was supplemented with 50 µL of FITC-12-dUTP and incubated at 37 °C for 1 h, followed
by testing using a machine. Triplicate experiments were performed independently.

Flow cytometry analysis of cell apoptosis
5
The U251 cells in the growth stage were seeded in 6-well plates at a density of 2.5 × 10 cells/mL, with 2 mL
per well. The drug (0, 0.1, 0.5, and 1 µmol/L) was added after cell adhesion. After a 24-hour incubation, the
cells were harvested using ethylenediaminetetraacetic acid-free pancreatic enzymes and washed twice with
PBS. 500 µL of 1× Annexin V Binding Buffer was added to each sample. Subsequently, 5 µL of annexin V-
FITC staining solution and 5 µL of PI staining solution were added to each sample, incubated at room
temperature for 5-15 min, and then analyzed by machine. Triplicate experiments were performed
independently.

RT-qPCR analysis
To investigate the mechanism of action of isocuB in glioma, we used RT-qPCR. First, RNA was extracted
from U251 cells treated with isocuB (0, 0.1, 0.5, 1 µmol/L) using TRIzol. RNA concentration and purity
were determined using a miRNA protein quantification assay. The assay was performed according to the
instructions provided with the SYBR® Green Real-time PCR Master Mix kit. Cycle threshold (Ct) was
determined using quantitative PCR. The primer sequences for MMP-2, MMP-9, PDK1, RXRα, PPARα,
Bcl-2, and β-actin are provided in Table 1. Additionally, we analyzed the RNA content of hsa-mir-1286a
using U6 as the internal reference. Hsa-miR-1286a inhibitor and microRNA inhibitor NC were then added
to the 6-well plate at a concentration of 100 µmol/L. RNA was extracted 24 h later, and the level of hsa-miR-
1286a was detected. Moreover, hsa-miR-1286a mimic was added to the 6-well plate at a concentration of
100 µmol/L. RNA was extracted 24 h later, and the level of hsa-miR-1286a was detected. Triplicate
experiments were performed independently.

WB analysis
To further investigate the mechanism of action of isocuB on glioma, the proteins in U251 cells treated with
different concentrations of isocuB (0, 0.1, 0.5, 1 µmol/L) for 24 h were extracted using RIPA lysis. The
extracted protein solution was then assayed using the BCA kit and denatured using SDS. Proteins were
separated at a constant voltage of 110 V for 90 min and then transferred to a PVDF membrane (Mannheim,
Germany). After blocking with 5% skim milk powder, the primary antibodies for MMP-2, MMP-9, p-PI3K,
PI3K, p-AKT(S473), AKT, p-STAT3, STAT3, RXRα, PDK1, p-MAPK1/3, MAPK1/3, N-cadherin, Vimentin
and Bcl-2 (1:1000) were added and incubated overnight at 4 °C. After washing with TBST, appropriate
secondary antibodies were added, incubated at 37 °C for 1 h, and then washed with TBST and TBS.
Luminescent agents were then added to facilitate detection in the chemiluminescence imaging system, and
further analysis was performed using ImageJ software. Triplicate experiments were performed
independently.

Establishment of U251/TMZ resistant strains
Cells in the logarithmic growth phase (80%-90%) were treated with drugs at a low initial concentration
(1/10-1/5 of IC of the parent cell line is recommended), and cultured in an incubator at 37 °C and 5% CO .
50
2
When the cell density reached 50%, the culture medium was abandoned, PBS was cleaned twice, and the
drug-free medium was replaced for further culture. When the cell growth density recovered to 80%-90%, the