Transwell kit

The Transwell kit (Gibco Company, USA) was used for detection of cell invasion. Cells cultured for 24 h were planted on a 6-well plate (5*10⁴ cells/well), washed twice with PBS, and inoculated in the upper chamber. The upper chamber was added with 200 μL of DMEM, while the lower chamber was added with 500 mL of DMEM (containing 20%FBS). After incubation at 37 ° C for 48 h, the matrix and cells not penetrating the membrane in the upper chamber were wiped off. The remaining cells were rinsed 3 times with PBS, fixed with paraformaldehyde for 10 min, rinsed another 3 times with double distilled water, then air dried and stained with 0.5% crystal violet. A microscope was employed to observe cell invasion. Cells cultured for 24 h were planted on a 6-well plate (2x10⁵ cells/well). After 24 h, a pipette tip was used to make a straight line along the diameter of the plate. Floating cells were removed lightly with PBS. Under a 20x microscope, 5 fields of view were randomly selected in each well for imaging. Then the cells were cultured in serum-free medium for 36 h, and the imaging was carried out in the same way as mentioned above.

The following are the sources of reagents, instruments, and cells: U87, T98G, U251, LN-229, and HEK-293T cells (Bena Culture Collection, China, BNCC337885, BNCC338721, BNCC100123, BNCC341218, and BNCC341976); Lipofectamine™ 2000 (Invitrogen™, USA, 11668019); a MTT assay kit and a dimethyl sulfoxide (DMSO) reagent (Beyotime Biotechnology, Shanghai, China, C0009, ST038); a Transwell kit, RPMI-1640, phosphate buffer solution (PBS), fetal bovine serum (FBS), and a penicillin-streptomycin double antibody (Gibco, USA, A1142802, 61870044, 14190250, 10437028, and 15070063); trypsin (Thermo Scientific™, 90058); TRIzol (Invitrogen, USA, 15596018); TransScript II Green Two-Step qRT-PCR SuperMix (TransGen Biotech, Beijing, China, AQ202-01 and AQ301-01); an Annexin V/PI apoptosis detection kit (Shanghai Yeasen Biotechnology Co., Ltd., 40302ES20); a microplate reader (BioTek, USA, PerkinElmer); a PCR instrument (ABI, USA, 7500); and a flow cytometer (BD, USA, FACSCanto II). Primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.