2.4. [3H]-Thymidine Incorporation Assay

Quiescent cells were treated with 10 ng/ml TNF-α and SMT, respectively, and 1 μCi of [³H]-thymidine was added (methyl-[³H] thymidine, 50 Ci/mmol; Amersham, Oakville, Ontario, Canada). After 24 h of incubation, cells were washed once with 2 ml of ice-cold PBS for 10 min, extracted three times with 2 ml of cold 10% TCA for 5 min each, and solubilized for at least 30 min at room temperature in 0.2 ml of 0.3 N NaOH, 1% SDS. After neutralization with 0.2 ml of 0.3 N HCl, [³H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA). Each experiment was conducted either in triplicate or in quadruplicate.