Cellular uptake study of DONs in H1299 and DMS53 cells. (a) Flow cytometry analysis of the uptake of DONs into H1299 and DMS53 cells after 0.5, 2, 4, and 8 h. Nontreated (NT) and Cy5-DNA treated cells were included as negative controls. Each column represents the average percentage of cells with Cy-5 positive fluorescence from three independent experiments. Error bars represent standard deviations. *P < 0.05 compared with NT, Cy5-DNA, ST, and SR. (b) Representative confocal microscopy images of DONs in H1299 cells after 8 h of incubation. Strong Cy5 fluorescence in the cytoplasm was observed for cells treated with DONs but not NT and Cy5-DNA controls. Blue: DAPI, red: Cy5. Scale bars: 25 μm. (c) Time-course study of ST uptake into H1299 cells by confocal microscopy. ST was found inside cells at as early as 5 min. Enhanced accumulation of Cy-5 fluorescence in cells was observed with increasing incubation time up to 72 h. Fluorescence was decreased after 96 h of incubation. Scale bars: 25 μm. (d) Uptake of ST and LR in both cell lines with inhibition of scavenger receptors. Significant inhibition (90% reduction) of ST and LR uptake into both H1299 and DMS53 cells was observed for cells treated with Poly-I, which binds and saturates scavenger receptors to block their accessibility to DONs. Each column represents the average percentage of cells with Cy-5 positive fluorescence from three independent experiments. Error bars represent standard deviation. *P < 0.05 compared against NT.