Fig. 2. Resistance to IR and cisplatin by increased BUB1B/BUBR1 expression in T24R and JMSU1R BC cells.

a Schematic representation of the protocol for the establishment of T24R and JMSU1R CRT-resistant BC cell lines. Parent T24 and JMSU1 BC cell lines were treated with IR (2 Gy/5 fraction × 10 cycle: total 50 Gy) and 2 µM of cisplatin. b Soft-agar colony-formation assay in T24, T24R, JMSU1, and JMSU1R cell lines treated with 1 µM of cisplatin. The medium was changed every 3days, and representative images after 21 days are shown. Lower panels exhibit immunoblotting in indicated cell lines. B-actin was loaded as an internal control. Quantitative evaluation by integrated optical density (IOD) for the immunoblotting was performed in three independent experiments, and the results are shown as mean + SD. *P < 0.05, unpaired t-test. c BC cell lines were treated with IR in the indicated dose, followed by the measurement of cell-viability assay after six days. The inhibitory effect on cell growth by the IR is presented as a relative value (mean ± SD) compared with control (0 Gy) as 100%. d Immunoblotting in indicated cell lines transfected with siRNAs three days before the IR treatment. Cells were collected 24 h after the IR treatment. B-actin was loaded as an internal control. e, f Cells were transfected with indicated siRNAs and incubated for three days. Thereafter, those cells were treated with or without 5 Gy of IR. Two days later, caspase 3/7 activity was measured. * indicates p < 0.05. g Left panel: cell-cycle analysis in T24 and T24R cells. Cell-cycle synchronization was performed by serum starvation in 0.1% FBS (G1 phase), 0.5 µM of etoposide (S phase), and 1 µM of nocodazole (G2/M phase) for 36 h. Right panel: Immunoblotting in T24 and T24R cell lines. Nuclear fractions incubated with indicated drugs for cell-cycle synchronization were subjected to immunoblotting with the indicated antibodies. Histone 3 was loaded as an internal control. h Immunoblotting of shControl and shBUB1B#1,2 in T24R and JMSU1R cell lines. Cells were cultured with 0.15 μg/ml of doxycycline for three days, then subjected to immunoblotting with indicated antibodies—right panel: Soft-agar colony-formation assay in these cells. Cells were cultured with 0.15 μg/ml of doxycycline for three days, then treated with IR (2 Gy/5fr every day). The medium was changed every 3days. Representative images are shown after 21 days. The number of colonies was counted in five random fields in 21 days, and the results are shown as mean ± SD. * indicates p < 0.05. i Schematic of the protocol for the xenograft mouse model. After tumors developed reaching 150 mm³ of tumor volume, mice were randomized into four groups with five mice in each group. j Tumor growth of T24R shBUB1B#1 and JMSU1R shBUB1B#1 cells in the xenograft mouse model treated with or without IR and doxycycline feeding. The result is shown as mean ± SD. k Representative images of γ-H2AX-positive foci induced by 5 Gy of IR in T24R sh-BUB1B#1 cells with or without 0.15 μg/ml of doxycycline. Scale bar indicates 10 μm. The bottom panels show the quantification of the number of γ-H2AX-positive foci among indicated cells cultured with 0.15 µg/ml of doxycycline. The results are shown as mean ± SD. * indicates p < 0.05. l Immunofluorescence of double staining with rH2AX and BUB1B/BUBR1 antibodies in T24R cells. Cells were treated with or without 6 Gy IR treatment, then fixed three hours after the treatment.