Figure 2. Resveratrol selectively causes cell cycle arrest in HNSCC cells independent of Smad4.
A) Cal27 cells were transfected with pcDNA Flag-Smad4M (Plasmid 14959) or vector control pcDNA3 (Addgene, Cambridge, MA) using Lipofectamine 2000. Stable transfectants were selected using G418 at 0.2 mg/ml in DMEM (10% FBS) for 4 weeks, and cells were pooled and maintained in the same selective medium. Cells lysates were prepared and subjected to SDS-PAGE followed by immunoblotting, and membranes probed with Smad4 antibody as detailed in Methods. Percentage cell cycle distribution in B) FaDu, C) Cal27-pcDNA3 and Cal27-Smad4, and D) NHEK cells following resveratrol treatment for 24 h at indicated concentrations and FACS analysis as detailed in Methods. Results shown are representative of triplicate samples from each treatment, and were reproducible in two independent experiments. NS, nonspecific; Res, resveratrol.