(A) Alterations in the signal transduction of MAPKs. HaCaT cells were incubated in medium containing everolimus at the indicated concentrations for 2 h after pretreatment with 10 μM stattic or DMSO. Total cell lysates were separated by SDS-PAGE and electrotransferred to PVDF membranes.

Various proteins and phosphorylation levels were evaluated by immunoblotting assay with specific antibodies. (B) Effects of MAPK inhibitors on everolimus-induced cell growth inhibition. HaCaT cells were incubated with medium containing everolimus at the indicated concentrations Palbociclib cell line for 48 h after pretreatment with U0126 (a MEK1/2 inhibitor, 10 μM) for 2 h, SB203580 (a p38 MAPK inhibitor, 10 μM) for 1 h, SP600125 (a JNK inhibitor, 20 μM) for 30 min, or DMSO (their solvent) for 2 h. Cell viability was determined by WST-8 colorimetric assay. *p < 0.01 Student’s t test compared with control (DMSO). Each bar represents the mean ± SD (n = 4). (C) Alterations in the signal transduction of STAT3 in the presence of MAPKs inhibitor. HaCaT cells were incubated in medium containing 30 μM everolimus for 2 h after pretreatment with 10 μM stattic for 20 min (st), 10 μM U0126 for 2 h (U), 10 μM SB203580 for 1 h (SB), 20 μM SP600125 for 30 min (SP) or DMSO (D). Total cell lysates were separated by SDS-PAGE and

electrotransferred to PVDF membranes. AZD6244 manufacturer Various proteins and phosphorylation levels were evaluated by immunoblotting assay with specific antibodies. Effects of STAT3 Y705F and STAT3C transfection on everolimus-induced cell growth inhibition in HaCaT cells STAT3C is a constitutively active STAT3 that dimerizes constantly by substituting cysteine residues for specific Thiamet G amino acids within the C-terminal loop of the STAT3 molecule [23], which resulted in the assembly of STAT3 in the nucleus of transfected cells (Figure 6B and C). Transfection of cells with STAT3 Y705F

had a tendency to enhance the cellular toxicity of everolimus compared with transfection with an empty vector, but STAT3C had a tendency to relieve, as shown in Figure 6A. Figure 6 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.