MTT proliferation assay

The proliferation assay was performed as previously described [26]. Briefly, the human epidermoid carcinoma A431 cells or human breast cancer MCF7 cells (1×10⁴ cells/well), with high and low expression levels of EGFR, respectively, were incubated overnight. On the following day, different concentrations of peptides (0, 2, 5, 10, 50, 100μg/mL) were added in 0.5% FBS media with 0.2μg/mL panitumumab or human IgG, and then incubated for 72h. All treatments were performed in triplicate. Numbers of live cells were determined using the MTT reagent by reading at 570 nm. The proliferation inhibition rate was calculated by the formula: (OD_{human IgG} – OD_{panitumumab+peptide}) ×100%/OD_{human IgG}.

To determine the effects of antibodies induced by Hsc70 fusion protein, MTT assay was also performed. A431, SW480, and MCF7 cells were plated in 96-well microtiter plate. On the following day, Antibodies isolated from immunized mice were added at increasing concentrations, and incubation was continued for 72h. The OD was detected as described above.