Cck 8

Cell viability was determined as described previously with slight revision [17 (link)]. Briefly, the differentiated HL60 cells were plated in 96-well plates at an initial density of 8000 cells/well in the presence of 100 ng/mL lipopolysaccharide (LPS, Millipore-Sigma, St. Louis, MO, USA) and EPA, in the form of free or liposomes, was supplemented to final concentrations of 40 and 80 µg/mL. The cells were further cultured in a humidified atmosphere containing 5% CO₂. After incubation of 20 h, 10 μL of the cell proliferation reagent CCK-8 (Promega, Madison, WI, USA) was added in every experimental well. 4 h later, the absorption at 490 nm was measured by a microplate reader (SynergyNeo, BioTek, Winooski, VT, USA). All experiments were performed in hex plicate.

Cell viability was quantified using a cell counting kit-8 (CCK-8) assay (Promega, Kumamoto, Japan). Approximately 0.7 × 10⁴ cells were seeded in each well of a 96-well plate containing 100 μL RPMI medium supplemented with 10% FBS, 100 μg/mL penicillin, and 0.25 μg/mL streptomycin and were cultured overnight. After 20 h of incubation, various concentrations of STK899704 were added to the wells and the cells were incubated further for the indicated time. Cell viability was analyzed by performing the CCK-8 assay according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Apollo LB 9110; Berthold Technologies GmbH, Bad Wildbad, Germany). Also, cell viability was assessed by the MTS dye reduction assay, which measures mitochondrial respiratory function. Lung cancer cells were seeded (7 × 10⁴cells/mL) in 100 μL medium/well in 96-well plates, incubated overnight, and treated with various concentrations of STK899704, as described in the figure legends. Cell viability was calculated by assessing MTS metabolism as previously reported (Kwon et al., 2016 (link)). In brief, media samples (100 μL) were removed and incubated with 100 μL of MTS-PMS mix solution for 1 h at 37°C. Optical absorbance was measured at 492 nm using an ELISA reader (Apollo LB 9110, Berthold Technologies GmbH).

Proliferation of HUMSCs was analyzed using cell counting kit-8 (CCK–8, Promega) according to the manufacturer’s protocols. HUMSCs were plated at a density of 2 × 10³ cells with 100ul medium into each well of a 96-well flat-bottom tissue culture plate (BD Falcon, San Jose, CA, USA),cultured for 4 h,24 h,48 h,72 h. CCK8 solution was added to each well and incubated in standard culture conditions for 1 h. The absorbance was analysed at 450 nm using a microplate reader (BioTek, Winooski, VT, USA), wells without cells as blanks. The results were expressed as mean absorbance in nanometers. All experiments were performed in triplicate.

The cell viability was assessed by (CCK8, Promega) as previously described (30 (link)). Stably expressing PCDH20 cells or vector were cultured in 200 μl of a 96-well plate (2,000 cells per well) with 10% FBS-contained 1,640 for 24, 48, and 72h. The cells were co-cultured in 100 μl of serum-free 1640 containing 10 μl CCK8 at 37°C for 2h. Microplate reader (Multiskan MK3, Thermo Fisher Scientific, Schwerte, Germany) was used to measure absorbance at 450 nm.

The siRNAs targeting MSMO1 and controls were obtained from RioBio. Sequences of effective siRNA are as follows:
siMSMO1-1: 5’- GAACAGACUCUCAGUAUAAdTdT-3’;
siMSMO1-2: 5’- GCUGUGGAAUAUGUAGAUUdTdT-3’.
Cells were transfected in triplicates with siRNAs pool at 10 nmol/L concentrations mixed with Lipofectamine RNAiMAX reagent (Thermo Scientific) on a 96-well plate according to the manufacturer’s reverse transfection protocol. Twenty-four hours after plating, cells were treated with PTX (0.05 μM), DOX (0.5 μM), or dimethylsulfoxide (DMSO, 0.02%). The viability was measured in 48 h using CCK-8 (Promega).