Fig. 4

HSP70 and CRT may play an important role in shikonin-induced ICD-derived proteins in treated tumor cells, that may differentially activate DCs and mediate T-cell proliferation. The shikonin-treated 4 T1 cells and the resultant-ICD derived TCLs (i.e., SK-TCL) and the specific protein-deleted SK-TCL [(i.e., SK-TCL(−HSP70), SK-TCL(−CRT) or SK-TCL(−HMGB1)] were used to stimulate DCs and for subsequent activation of T-cell proliferation. (a) Western blot analysis of ICD component proteins in SK-TCL(−HSP70), SK-TCL(−CRT) and SK-TCL(−HMGB1) samples, representing specific depletion of HSP70, CRT or HMGB1 proteins in the correspondent SK-TCL sample preparations, respectively. These DC vaccine samples were then used as stimulator cells for T-cell proliferation assays. Splenic CD4⁺ and CD8⁺ T cells were collected from syngeneic mice as responder cells. Proliferation activities of (b) CD4⁺ and (c) CD8⁺ T cells were analyzed by MLR assay. Ratios of stimulator to responder cells were set between at 1:1000 and 1:5. T-cell proliferation activity is represented as the fold change over the control (T cells only). Data represent the mean ± SD of three replicates. Similar results were obtained from three independent experiments