STAT3 and protein kinase B inhibition assay

In the in vitro studies, either cryptotanshinone (STAT3 inhibitor; TargetMol, Boston, MA) or ARQ 092 (protein kinase B [AKT] inhibitor; TargetMol) were dissolved in dimethyl sulfoxide and added to PBMC culture medium with a concentration of 10 μM or 500 nM, respectively. After 72 hours’ exposure, PBMCs were harvested for flow cytometry. For murine models, either cryptotanshinone or ARQ 092 was dissolved by using sterilized 0.5% carboxymethylcellulose sodium. SCID mice were treated with STAT3 inhibitor (20 mg/kg), AKT inhibitor (30 mg/kg), STAT3 inhibitor plus decitabine (0.03 mg/kg), AKT inhibitor plus decitabine, or decitabine. Inhibitor administration began from day 7 and was administered 3 times per week by gavage. After 3 weeks of administration, all mice were euthanized, and splenocytes were obtained for flow cytometry.