Fig. 4

VitK3-induced ROS accumulation in Prx1– cells. a–b Measurement of intracellular ROS level using carboxy-H₂DCFDA in Prx1+ and Prx1– HeLa cells (a) and in siPrx1- and siCon-treated HUVEC cells (b) after 15 μM vitK3 treatment. Results are plotted as cell counts versus fluorescence intensity. Gray shade, red line, green line, black dotted line and black solid line indicate the fluorescence intensity of the cells before treatment and after vitK3 treatment for 0.5, 1, 2 and 4 h, respectively. One set of three independent experiments is presented. c Fluorescence microscope and image analysis of cells expressing cyto-, nuc- and mito-HyPer. The time point before the addition of vitK3 is set as “0”. At upper panel, the upper and middle rows show acquired images under the YFPex/YFPem and the CFPex/YFPem channels. The lower row of images represents distribution of HyPer ratio. Scale bar = 6 μm. The color scale for the ratio values represents arbitrary unit of the spectrum, with less oxidized HyPer in green and highly oxidized HyPer in peak red. At lower panel, time course for the F/F0 ratio values (see Materials and Methods) in single cell expressing cyto-, nuc- or mito-HyPer during vitK3 treatment is presented. Fluorescence intensity of HyPer was recorded every 10 s over 5 min