Fig. 1

Structural requirements and energy-dependence for the uptake of Hst1. a HO1N1 cells were pre-incubated with 2 μmol·L⁻¹ F-Hst1 and its variants for 60 min, then nuclear DNA was stained with NucBlue™ live cell regent for 20 min. Representative a confocal laser scanning microscopy (CLSM) images of internationalization of F-Hst1, F-D-Hst1 and F-Hst1scr in HO1N1 cells. b Energy dependence experiments were performed by cooling the cells at 4 °C for 60 min to minimize cellular activity. Subsequently, cells were incubated with metabolic inhibitors 20 μmol·L⁻¹ NaN₃ (inhibitor of cytochrome C oxidase) and 50 μmol·L⁻¹ ouabain (inhibitor of Na⁺/K⁺-ATPase) at 37 °C for 60 min. Then cells were incubated with 2 μmol·L⁻¹ F-Hst1 for 60 min and stained with NucBlue™ live cell reagent for 20 min to stain DNA. Representative images of internationalization of F-Hst in HO1N1 cells at 4 °C cultured in medium with NaN₃ or with ouabain with CLSM Bar = 10 μm, F-Hst1 (in red); nuclei (in blue). c Mean intracellular fluorescence intensity of F-Hst1, F-D-Hst1 and F-Hst1scr was quantitated using Fiji. d Mean intracellular fluorescence intensity of F-Hst1 was quantitated using Fiji software. Data are presented as mean ± SD (n = 40); ****P < 0.000 1