scuPA, uPAT, and FV share an endocytic pathway in CD34⁺ MKs. (A) Representative confocal images of day-10 CD34⁺ MKs loaded simultaneously by preincubation with Alexa-568 scuPA (red) and Alexa-488 uPA-T (green) for 24 hours. Nuclear staining by DAPI is in blue. Overlap is in yellow and is shown to the right. Scale bar is shown. Quantitative analysis of overlap is shown in Table 1. (B-C) Similar confocal image studies as in panel A but of CD34⁺ MKs preincubated with (B) Alexa-488 scuPA or (C) Alexa-488 uPAT for 24 hours and then incubated with Alexa-568 FV for 2 hours (red) on day 11. White segmented lines in overlay images represent profiles along which the intensity of the fluorescence signal in each channel was measured using the ImageJ software. The plotted profiles are presented in right panels. Abscissa indicates the length of the profile in μm. Ordinate indicates relative fluorescence intensity. Coincidence of the peaks might provide clear evidence for colocalization of the signal in indicated channels. (D-E) Uptake of (D) Alexa-488 scuPA or (E) Alexa-488 uPAT, each at 400 nM, by day-11 CD34⁺ MKs in the absence or presence of Alexa-568-FV (0-400 nM). Abscissa denotes MFI measured by flow cytometry. Mean ± 1 SD are shown. n = 4 independent experiments.