- Cell Culture
The glioblastoma U-373 cell line was purchased from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 IU/ml penicillin, and streptomycin (pH 7.4) (all from Gibco BRL, Grand Island, NY), with 5% CO2 at 37°C.
- CCK-8
Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma, USA) according to the manufacturer's instructions. Cells were treated with 0.25 mmol/L caffeine, 0.5 mmol/L caffeine, 0.5 mmol/L caffeine, and FAK inhibitor (PF573228), respectively, for 24 h. CCK-8 was then added to the wells, and the assay was performed 1 h later. Absorbance was measured at 450 nm.
- Transwell assay
To detect cell migration ability, we used the transwell method. U-373 cancer cells in good growth condition were cultured in a 6-well plate, and when the cell density reached about 85%, cell transfection was performed. Then, based on the reference steps of the Transwell experiment, an invasion experiment was conducted to detect cell migration ability.
- Flow Cytometry Detection
U-373 single-cell suspension was inoculated onto 4 pieces of 12-well plates (10 × 104/well, 1ml/well) and cultured in an incubator for 24h. Once the cells adhered, 1 control group and 3 experimental groups were set (0.25mmol/L, 0.5mmol/L, and 0.5mmol/L + P13K/AKT inhibitor). The 12-well plate with added caffeine was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 24h. Early apoptosis of tumor cells was detected using the Annexin V + PI double staining flow cytometry method. Specific operation method: Take out the 12-well plate at the time point of caffeine action. Add 0.25% trypsin (200 μl/well) to each well to digest and make the cells suspended. Then add DMEM (1 ml/well) to neutralize the trypsin, make a cell suspension, and transfer it to a labeled tube. Centrifuge (1,000 g, 3 min), remove the supernatant, add DMEM (500 μl/tube), and blow into a cell suspension. Then transfer it to a labeled flow tube, successively add Media Binding Reagent 10 μl/tube, 1.25 μl Annexin V-FITC/tube, incubate at room temperature in the dark for 15 min. Centrifuge (1,000 g, 5 min), remove the supernatant, add 4°C 1 × Binding Buffer 0.5 ml/tube, blow into a cell suspension, add PI 10 μl/tube, and then detect on the machine. Test 1 × 104 cells for each sample. Obtain cells and analyze images using FlowJo software (Becton Dickinson).
- Cell Scratch Assay
This study used a cell scratch assay to assess cell migration ability. In brief, before inoculating the cells, three parallel lines were drawn at the bottom of each well of a sterile 6-well plate with a marker pen to locate the wound to be photographed. Cells in the logarithmic growth phase were inoculated into a 6-well plate at a density of 2×105 cells/ml and placed in an incubator for adherent growth. After the cells covered more than 95% of the bottom area of the plate, a wound was formed by scratching the cell monolayer with the tip of a 1 mL pipette. The wound and parallel lines drawn by the marker pen included several intersection points, and then the wound area crossing these lines was selected as the image capture location. Phosphate-buffered saline was used to remove floating cells, and the cells were cultured in a low-serum medium (1% fetal bovine serum) containing chemicals. Images of the selected wound area were obtained 0-24 hours after treatment under an inverted microscope (CKX41, Olympus, Japan) (600D, digital camera, Japan 600D). Six images were collected from each well at each time point and quantified. Cell migration was analyzed using Image-Pro Plus 6 software (Media Cybernetics, USA), and the reduced wound area in each image was divided by the wound length to calculate the migration distance.
- Western blot assay
After collecting cells and obtaining total protein, the protein concentration was determined using a BCA protein quantification kit. The protein concentration was analyzed using a BCA kit. Subsequently, the target protein was separated using SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk for 2 hours, and incubated with the primary antibody overnight at 4°C. The PVDF membrane was then incubated with specific primary antibodies: AKT (1:1000), P-AKT (1:500), PI3K (1:500), Bcl-2 (1:1000), Bax (1:1000), Caspase3 (1:1000), GPX4, ACSL4, and GAPDH (1:10000). After 24 hours, the protein was incubated with the secondary antibody (1:1000) at room temperature for 1 hour and then imaged using ECL reagent