Cell culture
U251 human glioblastoma cells were purchased from China Infrastructure of Cell Line Resource (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Tech, USA) supplemented with 10% fetal bovine serum (FBS; BI, Israel) and antibiotics (penicillin and streptomycin, each 100U/mL; Beijing Transgen, China). Cultures were incubated at 37°C in a humidified chamber with 5% CO2.
For sphere culture in vitro, U251 cells were seeded at 5000 cells/mL in six-well ultralow adherence plates (Corning Inc., USA) in serum-free DMEM/F12 (Life Tech) containing 20 ng/mL recombinant epidermal growth factor (Life Tech), 20 ng/mL basic fibroblast growth factor (Life Tech), B-27™ Supplement (Life Tech), and 1% penicillin/streptomycin (Beijing Transgen). The medium was changed every 48 h.
Cell viability assay
Cells were seeded in 96-well plates at 1 × 104 cells per well in 10% FBS-supplemented DMEM. The following day, the cell monolayers were treated with 12.5, 25, 50, 100, 200, or 400 µg/mL TMZ (Aladdin, China) alone or in combination with 1.25, 2.5, 5, 10, or 20 µg/mL CoAc2 (synthesized by our laboratory) for 24 h. MTT solution (5 µg/mL; Sigma, USA) was added (20 µL per well). After 4 h of continuous incubation, the supernatant was discarded, followed by the addition of DMSO (100 µL/well; Solarbio, China). The absorbance at 570 nm was measured. The Chou-Talalay method and CompuSyn software (version 1.0, ComboSyn, Inc., Paramus, USA) were used to detect the interaction between TMZ and CoAc2, which was quantified using a combination index (CI) as follows: CI < 1, synergism; CI = 1, additivity; and CI > 1, antagonism.
After the digestion of spheres in each group, the cells were resuspended in 500 µL of sphere medium, plated into 24-well ultralow adherence plates, and cultured for 4 h with 10 µL of Cell Counting Kit-8 (CCK-8) solution (TransDetect® Cell Counting Kit; Beijing Transgen). Then, 100 µL of culture medium were placed into each well of 96-well plates, and the absorbance at 450 nm was measured using a microplate reader.
Colony formation assays
Glioma cells were seeded into a six-well plate at a density of 1 × 104 cells per well, and the corresponding concentration of drugs was added after 24 h. The medium was changed every 2 days. After 10 days of culture, the cells were fixed with 75% alcohol, stained with crystal violet, dried, and counted.
Immunofluorescent staining
Cells were fixed in 4% paraformaldehyde (PFA) for 30 min, permeabilized in 0.5% Triton X-100 (prepared in PBS) for 20 min at room temperature, and incubated with mouse monoclonal anti-Nestin (1:600; Roche Diagnostics GmbH, Mannheim, Germany) overnight at 4°C. Cells were then incubated for 2 h with goat anti-mouse IgG H&L (Alexa Fluor® 488, 1:400; Abcam, UK), photographed, and observed using a fluorescent microscope.
Protein extraction and Western blot assays
Cells were seeded into a six-well plate and treated with the drugs for 24 h, followed by rinsing with ice-cold PBS solution. The cells were lysed with RIPA lysis buffer (Beyotime, China). The cell supernatant was collected by centrifugation at 12,000 × g for 5 min. SDS-PAGE sample loading buffer was added, followed by boiling at 100°C for 5 min. After SDS-PAGE electrophoresis, the total protein was transferred to a PVDF membrane (Millipore, USA), blocked with 5% skim milk for 2 h, and incubated using the primary antibodies overnight at 4°C. After three rinses in TBST, the membrane was incubated with secondary antibodies for 2 h and finally developed using ECL. ImageJ software was used to analyze and quantify protein expression. The primary antibodies included phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody, p44/42 MAPK (Erk1/2) (L34F12) mouse monoclonal antibody (mAb), MGMT (E6M7V) rabbit mAb (CST, USA), and GAPDH (0411) mouse mAb (Santa Cruz, USA). The secondary antibodies were anti-rabbit IgG (HRP-linked) antibody and anti-mouse IgG (HRP-linked) antibody (CST).
Transwell migration and Matrigel® invasion assays
U251TMZ cells were seeded in the upper compartment of 24-well Transwell plates at a density of 3 × 103 cells/well without FBS. For the invasion assay, 100 µL Matrigel® diluted at 1:3 in DMEM was placed in each well of the Transwell and incubated at 37°C for approximately 30 min for solidification before plating transfected cells. The lower compartment was filled with 600 µL DMEM supplemented with 10% FBS. After 48 h of incubation at 37°C in the presence of 5% CO2, the cells that did not migrate or invade and stayed on the upper surface of the filter were obliterated using a sterile cotton swab. The cells that migrated and invaded through the membrane into the bottom chamber were fixed with 75% ethanol for 30 min and stained with crystal violet. Images captured were used to count cells in randomly selected six fields per well using a microscope (DMI3000B, LEICA). The experiment was repeated three times.
RNA library construction and sequencing
U251TMZ cells were treated with 20 µg/mL CoAc2 or the same volume of DMSO as a control. The total RNA of the cells was extracted using TRIzol after 24 h. The samples were entrusted to GENEWIZ China & Suzhou Lab for subsequent RNA sample quality testing, ribosome removal, library construction, library purification, library detection, library quantification, and sequencing cluster generation. The samples were finally sequenced and analyzed on the Illumina HiSeq X Ten platform.
Statistical analysis
All experimental data were obtained through three independent experiments and presented as the mean ± SD. Student’s t‑test was used to calculate the statistical significance of the experimental results. p < 0.05 denoted statistical significance.