Dox-induced HMGB1 in MDA-MB-231 cells. (A) Intracellular HMGB1 expression was measured by real time PCR. Bars represent mean ± SD of HMGB1 expression level normalized against ACTB and relative to no drug treatment. Three independent experiments were performed. (B) Extracellular HMGB1 protein was detected by Western blot analysis in the culture media from cells treated with different concentrations of Dox for 48 h. (A and B) *p-value of less than 0.05 compared to the controls without Dox treatment. (C) BCF-CM induced resistance to Dox-mediated cell death in MDA-MB-231 breast cancer cells. Bars represent % cell death of each pre-treatment condition and the images show corresponding cell density. (D) Culture media from each condition in (C) were measured for extracellular HMGB1 by Western blot analysis. Equal amounts of proteins were loaded. Bars represent the mean band intensity (± SD) measured by densitometry. The band intensity of control cultures without CM pre-treatment (Ctl) was set as 1. *p-value of less than 0.05.