Annexin V and caspase 3/7 activity. Cells seeded on a 96-well plate (3,000 cells/well in 100 µl/well) in triplicates were allowed to adhere overnight. Annexin V reagent (Incucyte Annexin V Green Reagent for Apoptosis; Essen Bioscience, final dilution of 1:200) or Caspase 3/7 reagent (Incucyte Caspase 3/7 Green Apoptosis Reagent; Essen Bioscience, final dilution of 1:1,000) were added together with paclitaxel and/or EPO 72 h after cell seeding and EPOR silencing (experimental groups with siRNA and nt siRNA against EPOR). The rate of activation of Annexin V and both caspase 3/7 in cells was monitored with the Incucyte ZOOM system every 1 h after treatment of the cells. Plates were pre-warmed prior to data acquisition to avoid condensation and expansion of the plate, which would hinder autofocus. The maxima of excitation and emission were 490/515 nm and 500/530 nm for Annexin V and caspase 3/7, respectively. Images of wells of the 96-well plate were collected by Nikon 20x objective. Incucyte ZOOM integrated software (Essen Bioscience) was used to minimize background fluorescence and quantify fluorescent objects.
Statistical analysis. The data were statistically analyzed using ANOVA followed by Tukey's multiple comparison tests in ORIGIN analysis software (OriginLab Co., Northampton, MA, USA). The results were considered significant at the probability level P<0.05 and P<0.01.
Statistical analysis. The data were statistically analyzed using ANOVA followed by Tukey's multiple comparison tests in ORIGIN analysis software (OriginLab Co., Northampton, MA, USA). The results were considered significant at the probability level P<0.05 and P<0.01.