Cells were seeded onto 96 well plates and precultured for 24 h. The medium was exchanged for medium containing everolimus at various concentrations after pretreatment with signal transduction inhibitors at several concentrations, for appropriate term, followed by incubation for 48 h at 37 C. The culture medium was replaced with a medium containing a WST 8 reagent for 3 h and the absorbance in the well was deter mined at 450 nm with a reference wavelength of 630 nm using a microplate reader. Apoptosis assay Apoptosis mediated cell death was examined in HaCaT cells by a double staining method using a FITC labeled Annexin V propidium iodide apoptosis detection kit according to the man ufacturers instructions.

In brief, control, everolimus treated, and stattic treated cells were washed in phosphate buffered saline twice and incubated with PBS containing FITC conjugated Annexin V and PI dyes for 30 min at 37 C. After cells were washed in PBS twice, they were incubated with PBS containing 10 uM Hoechst 33258 and 4% para formaldehyde for 30 min at 37 C. The externalization of phosphatidylserine additional info and the permeability to PI were evaluated using an IN Cell Analyzer 2000. Cells in early stages of apoptosis were positively stained with Annexin V, whereas cells in late apoptosis were positively stained with both Annexin V and PI. Western blotting Western blotting was performed as described previously. Proteins in the total cell lysate were extracted from cells treating to each buffer with Cell Lysis Buffer in addition to 1 mM dithiothrei tol, 1 mM phenylmethylsulfonyl fluoride, and 5 ug mL leupeptin.

Proteins were separated order SH-4-54 using 7. 5 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane. Subsequently, the blot was blocked in a solution of wash buffer containing 5% skim milk. The membrane was soused in wash buffer containing specific primary antibodies overnight, followed by incubation with horseradish peroxidase conjugated secondary antibodies for 1 h. Antibody bound proteins were visualized by treat ing the membrane with the enhanced ECLTM Prime Western Blotting Detection Reagent pre pared immediately before detection. Finally, blot im ages were acquired using ChemiStage 16 CC. Wherever indicated, the membranes were stripped and reprobed with another antibody.

Plasmid construction Constitutively active STAT3 mammalian ex pression plasmids were kindly provided by Professor Miyajima. Tyro sine 705 deficient STAT3 mammalian expression plasmids were kindly provided by Darnell. STAT3C and STAT3 Y705F constructs were transformed into DH 5 competent cells and plasmid DNA was extracted using the QIAGEN Plas mid Midi Kit. Extracted plas mids were purified to a grade appropriate for cell culture using phenol and chloroform and stocked at 1 ug uL in a freezer until experimental use.