Figure 5

Effects of everolimus on MAPKs activation in HaCaT and effects of MAPK inhibitors on everolimus-induced cell growth inhibition and signal transduction. (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 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. Various proteins and phosphorylation levels were evaluated by immunoblotting assay with specific antibodies.