Cell activity was monitored using a cell counting kit-8 (CCK-8) (Dojindo, Shanghai, China) according to the manufacturer’s instruction as our previously described39 (link). HEK293 cells were pretreated with or without exogenous ETHE1 (CUSABIO) (100 ng/ml) or ACY-3 (DAKEWE, Bejing, China) (100 ng/ml) for 0.5 h and then incubated in the absence or presence of DU (500 μM) for 24 h. The CCK-8 solution (10 μL) was added to each well (100 μL) followed by incubation for 1 h. The absorbance was measured at 450 nm by a microplate reader (BioRad). The cell viability is expressed as the percentage relative to the control cultures. In addition, a Calcein-AM and PI double staining kit (Dojindo) was used for the double staining and analysis of the living cell and dead cell levels as our previously described39 (link). The living cells (yellow-green fluorescence) and dead cells (red fluorescence) were measured by a fluorescence microscope (IX51, Olympus, Tokyo, Japan) with a 490 nm excitation filter.
To further clarify the role of ETHE1 in MT protection from DU, cells transfected with or without ETHE1-specific siRNA or control siRNA were pretreated with or without exogenous MT (Sigma-Aldrich, Santa Clara, CA, USA) (100 nM) for 24 h and then incubated in the absence or presence of DU (500 μM) for 24 h. Cell viability was also determined by a CCK-8 assay.
To further clarify the role of ETHE1 in MT protection from DU, cells transfected with or without ETHE1-specific siRNA or control siRNA were pretreated with or without exogenous MT (Sigma-Aldrich, Santa Clara, CA, USA) (100 nM) for 24 h and then incubated in the absence or presence of DU (500 μM) for 24 h. Cell viability was also determined by a CCK-8 assay.