Fig. 2

In vitro fluorescence lifetime measurement of doxorubicin binding to chromatin. a–c diagrams of possible interactions between doxorubicin and H1-EGFP chromatin. a doxorubicin (yellow dots) intercalation into DNA (blue regions represent the core histone optima and dark blue dashed lines represent DNA) brings it close to histone H1-EGFP, leading to energy transfer (orange stars). b GFP is not fused to histone H1 and is therefore not sufficiently close to DNA bound doxorubicin for energy transfer to occur. c doxorubicin is not binding DNA and therefore no energy transfer with histone H1-EGFP occurs. d in vitro multiphoton LSM fluorescence lifetime images of IGROV-1 cells transfected with the indicated constructs and exposed to 9 μM doxorubicin (scale bar is 210 µm). e Box plots of mean fluorescence lifetime values per cell corresponding to fluorescence lifetime images in d. Red and blue box plots represent measurements with and without incubation with doxorubicin for 3 h, respectively, for each construct. On each box plot, the central mark indicates the median, and the bottom and top edges of the box indicate the interquartile range (IQR). The box plot whiskers represent either 1.5 times the IQR or the maximum/minimum data point if they are within 1.5 times the IQR. For each construct, the data represented by the red and blue box plots were compared with an unpaired, unequal variances two-tailed t-test and the associated P-values are shown above each pair of box plots as described in the Methods section. With n = 1 biological replicates, the number of cells imaged for each expression type with & without (w&wo) DOX was 6&10 for cells expressing free EGFP, 12&30 for cells expressing H1-EGFP, and 8&6 for cells expressing H2-EGFP. Imaged cells for each construct expression and with&without DOX combination were sampled from a single well of a 6-well plate and all six combinations were seeded on the same 6-well plate