Western Blot Quantification of Key Signaling Proteins

The Western blot procedures were performed as reported previously^{17 (link), 71 (link)}. Briefly, after determining the protein concentrations by the Bio-Rad protein assay. The cell lysates containing equal concentration of protein were solubilized in SDS-sample buffer and separated on SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against phosphor- and total Stat3, DNMT1 and EZH2 (1:1000 and 1:2000, respectively). The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase, and transferred to freshly made ECL solution (Immobilon Western; Billerica, MA, USA), followed by observing the signals under the Molecular Imager ChemiDoc XRS Gel Imagine System (BioRad, Hercules, CA, USA) and documenting the results.