M2009pr

Cell proliferation was examined by MTT assay. Cells (2,000 cells/ml) were seeded into 96-well plates and incubated at 37°C for 24, 48, 72, 96 or 120 h. At 4 h prior to each time point, 0.5% MTT solution (Thermo Fisher Scientific, Inc.) was added, followed by incubation for 4 h at 37°C. The cell supernatants were discarded, and the formazan crystals were dissolved in 100 µl dimethyl sulfoxide. The optical density (OD) of each group was measured using a microplate reader (M2009PR; Tecan Group, Ltd.) at a wavelength of 490/570 nm.

An MTT assay was applied to analyze in vitro cell viability. Following the trypsinization of cells in the logarithmic growth phase, glioma cells (2,000 cells/well) were seeded into a 96-well (100 µl/well) (cat. no. 3599; Corning Inc.) overnight. MTT solution (20 µl/well, 5 mg/ml; cat. no. JT343; Genview) was added to cells and incubated for 4 h. DMSO (100 µl/well) was added to dissolve the formazan crystals. Absorbance values were measured at 490 nm using a microplate reader (cat. no. M2009PR; Tecan Group, Ltd.) after 24, 48, 72, 96, and 120 h of growth; 570 nm was used as the reference wavelength. The cell viability ratio was calculated using the following formula: cell viability (%) = OD (treated)/OD (control) ×100%.

Apoptotic cells were assessed using the Annexin V apoptosis kit (88–8007; eBioscience). In short, cells were trypsinized, washed and centrifuged at 283 × g for 5 min. Then, 1X binding buffer was used to wash the cells again by resuspending the cells in 200 µl. Then, the cells were incubated with 10 µl Annexin V-APC at room temperature for 15 min in the dark. Finally, the Annexin V-stained cells were analyzed with a flow cytometer (Millipore) to determine the proportion of apoptotic cells.
Caspase-3/7 are central effector caspases in apoptosis and are usually used to measure apoptotic activities. GBM cells were first transfected with the ZWINT shRNA and NC. Next, 1×10⁴ infected cells/well were seeded in a 96-well plate. Caspase-Glo 3/7 reagent (100 µl, G8091; Promega) was added to each well, the plate was shaken for 30 min constantly and incubation was carried out at ambient temperature for 2 h. The luminescence signal was detected with an M2009PR (Tecan Infinite) plate reader.

After infection with lentiviruses expressing sh-MTHFD1L or scrambled shRNA, OSCC cells were cultured for 48 h and harvested during the logarithmic phase. Next, cell suspensions were generated and seeded in triplicate in 96-well plates at a density of 1,000 cells per well. After culturing at 37 °C with 5% CO₂ for an additional 24 h, images of the cell were captured daily for 5 d using a Celigo image cytometer system (Nexcelom; Lawrence, MA, USA). Cell numbers per well were also quantified using the Celigo system, and cell growth curves were plotted for each condition.
The cell viability was assessed using the MTT (Genview, Beijing, China) assay. Briefly, 1,500 shRNA-treated or untreated cells per well were seeded in 96-well plates. After 24 h at 37 °C, MTT (5 mg/mL) was added to each well before the terminal point of cell culture, and cells were incubated for an additional 4 h. Then, the medium was removed and formazan crystals were dissolved in 100 μL dimethyl sulfoxide (DMSO). The cell viability was determined by measuring the absorbance of each well at 490/570 nm and using a multi-well plate reader (Cat. #M2009PR; Tecan Infinite).