Figure 1
Assessment of DNR uptake in cancer cell lines.
After addition of DNR (5 μM) to cells, samples were harvested at the indicated time to monitor for the level of drug uptake. (A) Chemical structure of daunorubicin (DNR) obtained from https://www.ncbi.nlm.nih.gov/pccompound?term=daunorubicin. (B) Comparison of the relative uptake of DNR into four cancer cells using FACS analysis. Once the drug uptake was stopped, the washed cells were processed for FACS analysis. A total of 10,000 cells were analyzed for DNR fluorescence using FL2 (585/42). Results are expressed as the mean ± S.D. from three separate experiments. (C) Fluorescent spectrometry analysis of DNR uptake into TOV2223G, OV866(2) and HL60 cells. The control (Ctrl) are TOV2223G cells without DNR. Increased fluorescence denoting drug uptake was detected with Fluoroskan Ascent Microplate reader set at 544 nm excitation/590 nm emission. The relative fluorescent unit (RFU) representing the measurement of drug uptake was expressed as pmol DNR/2 × 104 cells/min (see Material and Methods). Results are expressed as the mean ± S.D. from three separate experiments. (D) Comparison of the concentration-dependent uptake of DNR by TOV2223G and HL60 cells. Cells were incubated with indicated concentrations of DNR then analyzed by the Fluoroskan Ascent Microplate reader. Results are expressed as the mean ± S.D. from three separate experiments. (E) Epifluorescent microscopy showing intracellular colocalization of DAPI and DNR in the TOV2223G cell line. Comparisons were made between TOV2223G cells treated with or without DNR, followed by staining of nuclear DNA. Following DNR uptake (5 μM), the cells were washed, fixed with paraformaldehyde and processed for microscopy using Vectashield mounting medium containing 1.5 μg/ml DAPI to detect the nuclear DNA. Images were captured with an Olympus BX51 epifluorescent microscope using Texas Red and DAPI-UV filters at 100× magnification and then processed with ImageJ software. Merged images overlapped DAPI-stained nucleus (blue) with DNR staining (red). Results are representative of two separate experiments.
