Cck 8 solution

For Cell Counting Kit-8 (CCK-8) assay, MDA-MB-231 cells (1×10³ cells/well) were seeded in 96-well plates and cultured for 24 h. At 24, 48 and 72 h after incubation, CCK-8 solution (Merck KGaA) was added to each well. The cells were incubated for an additional 1 h to determine cell viability. Optical density was determined at 450 nm using a microtiter plate reader.
For colony formation assay, transfected MDA-MB-231 cells (1×10⁵ cells/well) were seeded into 6-well plates, and the MDA-MB-231 cells treated with sh-NC or pcDNA3.1 were used as the negative controls. Following an incubation period of 14 days at 37°C, the colonies were fixed with 100% methanol for 15 min at room temperature and stained with 0.1% crystal violet at room temperature in absolute ethanol for 15 min. Visible colonies of >50 cells were counted and analyzed under an inverted light microscope.

Following transfection, HPrEC, 22Rv1 and DU145 cells were collected, washed with PBS and resuspended in RPMI-1640 medium (catalog no. 30-2001; ATCC) to the density of 3×10⁴/ml. Cell proliferation was measured by CCK-8 assay. The highly water-soluble tetrazolium salt WST-8 is reduced by cellular dehydrogenases, which becomes orange in color (formazan); the amount of formazan is proportional to the number of living cells. Cells were cultured in a 96 well plate at a density of 3×10³ cells/100 µl/well and placed in the incubator. CCK-8 solution (10 µl; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added at 24, 48, 72 and 96 h. Cells were then cultured for additional 4 h and 10 µl DMSO was added. OD values were measured at 450 nm using Fisherbrand™ accuSkan™ GO UV/Vis microplate spectrophotometer (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate. At 24, 48, 72 and 96 h, cells were subjected to RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to confirm the overexpression of GASL1.

Approximately 1 × 10⁴ OGD transfected or un-transfected HBMVECs were treated with or without OGD incubation, and then seeded into 96-well plates. Cells were cultured for 24 h, and 10 μL of CCK-8 solution (Sigma-Aldrich) was added and incubated for another 2 h. The optical density (OD) was detected by a microplate reader (Bio-Tek, Winooski, USA) at an absorbance of 450 nm.

CCK-8 assay was performed to assess cell viability. SGECs were seeded into 96-well plates (3x10³ cells each well). Following transfection for 24 h at 37˚C, cells were incubated with CCK-8 solution for 1 h following the manufacturer's protocols at 37˚C (10 µl each well; Sigma-Aldrich, Merck KGaA) before viability was subsequently analyzed at a wavelength of 450 nm, using a microplate reader.

To study the function of SGK1 on cell proliferation, the cells at the density of 5×10³ cells /well were seeded in a 96-well plate of y13B (LIU YI, Beijing, China; AF2006, Affinity). Vector construction and cell transfection or cells were treated with cisplatin (MB1055, meilunbio, Dalian) at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, and 6 μM after cell transfection. After further growth for different durations, 10 μL CCK-8 solution (96992, Sigma, USA) per well was added. After treatment at 37 °C for 1 h, the absorbance value at 450 nm was detected by a microplate reader (800Ts, BioTek, USA).