Effects of dominant negative and constitutively active STAT3 on everolimus-induced cell growth inhibition in HaCaT cells. (A) Effects of STAT3 Y705F and STAT3C transfection on everolimus-induced cell growth inhibition. HaCaT cells transiently transfected with STAT3 Y705F, STAT3C or each empty vector were incubated in medium containing everolimus at the indicated concentrations for 48 h after preincubation for 24 h. Cell viability was determined by WST-8 colorimetric assay. *p < 0.01 Student’s t test compared with control (DMSO). There was no significant difference in the cell toxicity between the empty vector and STAT3C transfection. (B) Immunostaining images. HaCaT cells transiently transfected with STAT3C or empty vector were fixed and incubated with an anti-Stat3 antibody, followed by incubation with FITC-conjugated anti-rabbit IgG (green) and visualization on an IN Cell Analyzer 2000. (C) STAT3 nuclear entry was determined by measuring the nucleus/cytoplasm intensity ratio of green fluorescence (n = 3). *p < 0.05 Student’s t test compared with control.