Approximately 5,000 cells were plated in 96-well plates with 200
μl medium per well. The cells were then transfected as indicated with sh-MSI1 (MSI1 suppression plasmid), negative control vector (pcDNA 3.1 or sh-Ctrl) or MSI1 overexpression plasmid (MSI1) for 48 h, after which time 2
μl
CCK-8 solution (Biosharp) was added to each well and the cells were incubated for a further 4 h. The absorbance at 450 nm was measured using a
micro-plate reader (Thermo Fisher Scientific, Inc.).
To detect
BrdU incorporation, the cells transfected with the indicated plasmids were washed thoroughly with medium and then cultured in fresh medium containing 10
μM
BrdU (Sigma-Aldrich; Merck KGaA) for 1 h. The cells were then allowed to grow in
BrdU-free medium for another 48 h, after which time they were harvested for detection. The harvested cells were washed with PBS, fixed in 70% ice-cold ethanol, resuspended in 2N HCl and incubated for 30 min at room temperature, followed by hybridization with a mouse monoclonal anti-
BrdU antibody (cat. no. ab8152, dilution, 1:500, Abcam) overnight at 4°C. Finally, the cells were rinsed with PBS combined with Tween-20 and incubated with
fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin antibody (Jackson ImmunoResearch), followed by staining with 4′,6-diamidino-2-phenylindole solution for 10 min prior to image capture.
Wang C.F., Zhang H.C., Feng X.M., Song X.M, & Wu Y.N. (2019). Knockdown of MSI1 inhibits the proliferation of human oral squamous cell carcinoma by inactivating STAT3 signaling. International Journal of Molecular Medicine, 44(1), 115-124.
