Cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cells
were seeded at 1 × 104 cells/well on a 96-well microporous plate and
incubated at 37°C with 5% CO2 for 0, 24, 48, 72 or 96 h. The medium
was renewed with 100 µL growth medium and 10 µL CCK-8 solution (MedChemExpress,
Monmouth Junction, NJ, USA) for another 3-h incubation at 37°C. The optical
density (OD) value at 450 nm was measured by a spectrometer reader (SpectraMax
M2, Molecular Devices, San Jose, CA, USA).
The effect of miR-214, VEGFA or Bcl-2 overexpression on cell proliferation was
also determined by Click-iT Plus 5-Ethynyl-2’-deoxyuridine (EdU) Alexa Fluor
1594 Imaging kits (Thermo Fisher). Transfected A431 and SCC13 cells were
immobilized with 50 µL cold 4% formaldehyde for 30 min at room temperature.
4’,6-diamidino-2-phenylindole (1:2000) was used to stain the nucleus at room
temperature for 30 min, and the signals were observed using Olympus FLUOVIEW
FV1000 confocal laser-scanning microscope (×100 magnification, Olympus
Corporation, Tokyo, Japan).
were seeded at 1 × 104 cells/well on a 96-well microporous plate and
incubated at 37°C with 5% CO2 for 0, 24, 48, 72 or 96 h. The medium
was renewed with 100 µL growth medium and 10 µL CCK-8 solution (MedChemExpress,
Monmouth Junction, NJ, USA) for another 3-h incubation at 37°C. The optical
density (OD) value at 450 nm was measured by a spectrometer reader (SpectraMax
M2, Molecular Devices, San Jose, CA, USA).
The effect of miR-214, VEGFA or Bcl-2 overexpression on cell proliferation was
also determined by Click-iT Plus 5-Ethynyl-2’-deoxyuridine (EdU) Alexa Fluor
1594 Imaging kits (Thermo Fisher). Transfected A431 and SCC13 cells were
immobilized with 50 µL cold 4% formaldehyde for 30 min at room temperature.
4’,6-diamidino-2-phenylindole (1:2000) was used to stain the nucleus at room
temperature for 30 min, and the signals were observed using Olympus FLUOVIEW
FV1000 confocal laser-scanning microscope (×100 magnification, Olympus
Corporation, Tokyo, Japan).