Expression of damage-associated molecular patterns (DAMPs) and tumor-associated antigens in tumor cell lysates of treated B16 melanoma. (A) The chemical structures of microtubule-depolymerizing agents and doxorubicin tested in this study are shown in A. (B) Effect of MDAs and doxorubicin on cell viability of B16 melanoma cells. Mouse B16 melanoma cells were treated with the indicated concentrations of colchicine, doxorubicin (DX), 2-(3-chlorophenyl)-6,7-methylenedioxyquinolin-4-one (CMQ) and 2-(3-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (FMQ) for 24 h, as shown in B. The values are represented as the percentage of viable cells; the vehicle control group was regarded as 100% viable. Cell viability was determined by MTT assay and data were expressed as mean ± S.D. for triplicate culture samples. (C&D) Effect of MDAs and doxorubicin on expression of damage-associated molecular patterns (DAMPs) and tumor-associated antigens in treated B16 melanoma cells. B16 tumor cells were treated for 24 h with vehicle control, colchicine (C), CMQ, FMQ or DX, at a concentration of 2.5 μM. After treatment, TCLs were obtained through four freeze-thaw cycles. Western blot analysis for protein expression of damage-associated molecular patterns (DAMPs), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), calreticulin (CRT) and high-mobility group box-1 (HMGB1), is shown in C, and expression of tumor-associated antigens including glypican-3 and survivin, is shown in D. Expression of β-actin was used as an internal control. The results show one representative experiment of three independently performed experiments.