Fig. 2

Analysis of γH2AX, 53BP1, and intracellular accumulation of doxorubicin in bat, human, and mouse cells. a Dose titration of doxorubicin (Dox) in PaLung, WI-38 and MEF cells. Cells were treated with the indicated doses of doxorubicin for 3 h, and protein lysates were analysed by Western blotting. Tubulin was used as a loading control. b Western blot analysis of γH2AX in PaLung, WI-38 and MEF cells treated with 5 μM Dox for 3 h, followed by drug-free medium up to 12 h (starting at t = 0 h, indicated by arrow). Protein lysates were harvested at the indicated time points. Tubulin was used as a loading control. N.T stands for no treatment. c Analysis of the average number of 53BP1 foci per cell for PaLung, WI-38 and MEF cells. Cells were treated with 5 μM Dox for 3 h, followed by drug-free medium up to 12 h. Immunofluorescence staining of 53BP1 was performed at the indicated time points. The number of foci in a minimum of 100 cells was quantified. N.T stands for no treatment. d The volume of the indicated cell lines. e Doxorubicin accumulation in PaLung, WI-38 and MEF cells after 3 h of incubation at the indicated concentration. The amount of accumulated doxorubicin in cells was analysed by flow cytometry. The mean fluorescence value (fv) of doxorubicin is normalised to cell volume (pL). f Time course of doxorubicin accumulation in PaLung, WI-38, and MEF cells. Cells were treated with 5 μM doxorubicin for the indicated time and analysed by flow cytometry. The mean fluorescence value of doxorubicin accumulated in each cell line is normalised to cell volume. All Western blot results shown are representative of at least three experimental repeats. Bars represent the means ± SDs of at least three independent experiments. Statistical significances were calculated using unpaired student’s two-sided t test. p < 0.05 is represented with *, p < 0.01 with ** and p < 0.001 with ***