Alexa fluor 488 goat anti rabbit and mouse igg

Cells on 8-well chamber slides (BD Biosciences) were fixed with 4% PFA, washed with PBS, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with primary antibodies in antibody diluent (Dako, Glostrup, Denmark) overnight at 4°C and then with Alexa Fluor-488 goat anti-rabbit and mouse IgG (Invitrogen) secondary antibodies. Cells were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired using a Carl Zeiss confocal microscope (Weimar, Germany).

Salinomycin, S3I-201, interleukin-6 (IL-6), paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), Triton X-100, and phosphate buffered saline (PBS) tablets were obtained from Sigma-Aldrich (St. Louis, MO, USA). LLL12 was purchased from BioVision Inc. (Milpitas, CA, USA). The 2 mM stock solution of Salinomycin was dissolved in DMSO and used at final concentrations of 0.5–10 μM. The 20 mM stock solution of S3I-201 and 1 mM stock solution of LLL12 were dissolved in DMSO and used at final concentrations of 50 μM and 1 μM, respectively. An equal volume of DMSO was added to control groups and the final concentration of DMSO was less than 0.5%. Aliquots of all stock solutions were stored at -20°C. Phosphatase inhibitor and protease inhibitor cocktail tablets were purchased from Roche Applied Sciences (Penzberg, Germany). Primary antibodies were used for the following proteins: STAT3, phospho-STAT3 (Tyr705) (Abcam, Cambridge, MA, USA); PARP, cleaved PARP, cleaved caspase-3, cleaved caspase-8 (Cell Signaling Technology, Beverly, CA, USA); cyclin D1, survivin, Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); actin (Sigma-Aldrich). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit and mouse IgG (Bio-Rad Laboratories, Hercules, CA, USA) and Alexa Fluor-488 goat anti-rabbit and mouse IgG (Invitrogen, Carlsbad, CA, USA).