Combination treatment with TMU-35435 and IR causes autophagic cell death. (A) Measurement of acidic vesicular organelles (AVOs) in 4T1 cells by flow cytometry. The cells were treated with TMU-35435 (1 μM) and IR (4 Gy) for 24 h. (B) Quantification of AVOs in acridine orange (AO)-stained cells treated with TMU-35435 and IR separately or in combination by flow cytometry in MDA-MB-231 and 4T1 cells. * p < 0.05, TMU-35435 versus combination treatment. # p < 0.05, IR versus combination treatment. (C) LC3-I and II expression using Western blot analysis. (D) Quantification of AVOs in AO-stained cells pretreated with 3-MA by flow cytometry. 4T1 cells were pretreated with 3-MA for 1 h before receiving the combination treatment. ** p < 0.05, combination treatment versus control. # p < 0.05, combined treatment +3-MA versus combined treatment. (E) The cytotoxicity was measured after cells were incubated with or without 3-MA. * p < 0.05, combination treatment versus control. # p < 0.05, combined treatment +3-MA versus combined treatment. (F) Western blotting of LC3-I and LC3-II expression in 4T1 cells. The cells were pretreated with BAF for 1 h and then treated with TMU-35435 (1 μM) and IR (4 Gy) for 24 h.