Effect of recombinant HMGB1 (rHMGB1) on the response of MDA-MB-231 to Dox. (A) MDA-MB-231 human breast cancer cells were treated with 5 μM Dox with or without addition of 100 ng/ml rHMGB1 and/or anti-HMGB1 neutralizing antibody for 24 h. The % cell viability is shown and the bars represent the mean ± SD of triplicate experimental wells. (B) Autophagy-related protein LC3B is induced in MDA-MB-231 breast cancer cells. Densitometric analysis of LC3B-II normalized against the protein loading control β-actin and the conversion of LC3B-I to LC3B-II is shown. (C) MDA-MB-231 cells treated with ‘dead cancer-CM’ showed increased viability compared with controls and this effect was attenuated by anti-HMGB1 neutralizing antibody. Bars represent mean ± SD of three independent experiments. * = p-value of less than 0.05 whereas ** is less than 0.01.